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Filling a Critical Need by Establishing a Fully Functioning, CODIS

1. Average PHR xX X Minimum PHR Figure 24 Peak height ratio summary results 85 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice AB 3500 ID Plus Validation Non Probative Evidence Summary Introduction Using actual convicted offender buccal DNA collectors from the CODIS archive at the Wyoming State Crime Laboratory 1 2mm punches were taken with the semi automated BSD 600 Duet placed into PCR setup amplified post amp prepared genotyped on a 3500 genetic analyzer and analyzed in GeneMapper ID X software At the time of this validation study the ultimate goal for this procedure is to establish a highly efficient and rapid method to process and genotype convicted offender samples Because the end purpose of the system exactly matches the substrates samples that have been used in the validation procedure the QAS criteria requesting authentic case samples has been met to the best of the Wyoming State Crime Laboratory s abilities Notes Pre 2006 convicted offender database samples are an archive of blood stains on FTA cards At this time there is also discussion regarding the move toward an indicating FTA paper buccal sample collector due to the potential of arrestee legislatio
2. 500 00 400 00 300 00 200 00 100 00 Figure 27 Peak height increases with Prep n Go buffer larger fragment loci are yellow intermediate green and smaller blue Average Peak Height Ratio Change with Prep n Go vs PunchPrep 35 00 30 00 25 00 20 00 15 00 10 00 5 00 0 00 5 00 OY Figure 27 Peak height ratio changes with Prep n Go buffer as a quality indicator larger fragment loci are yellow intermediate green and smaller blue 100
3. Stutter peaks are artifacts of the amplification process These peaks have a significantly lower RFU and typically are located one repeat unit n 4 before the true allele Stutter peaks may also appear at positions one repeat unit longer n 4 and two repeat units shorter n 8 than the true allele although not as commonly Consult the validation study for details on different types and combinations of stutter observed with the Identifiler Plus chemistry The following table should be used as a guideline for stutter peak height ratios reference ID Plus 3500 validation study n 4 cutoff Locus ratio D8S1179 10 45 D21S11 13 90 D7S820 9 69 CSF1PO 9 20 D3S1358 14 84 THO1 6 95 D13S317 9 93 D16S539 11 53 D2S1338 12 44 D19S433 11 67 47 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice vWA 13 65 TPOX 6 38 D18S51 14 96 D5S818 10 06 FGA 13 36 Peaks n 4 that are less than the cutoff ratio shall be considered stutter Peaks n 4 that are greater than the cutoff ratio shall be considered true alleles in the absence of other confounding factors v Rare variants gt Rare variants microvariants microheter
4. Figure 13 Precision Study results of Identifiler Plus on 3500 genetic analyzer AB 3500 ID Plus Validation Reproducibility Study Summary Introduction Allelic ladders for the Identifiler Plus kit were injected on the AB 3500 and analyzed with GeneMapper ID X software to determine the reproducibility of the current procedure being validated for the 3500 instrument Methods Two different allelic ladder plates were prepared according to the manufacturer s protocols for the formamide amplicon LIZ 600 v2 preparation The plates were created on different days 03 28 2011 and 04 05 2011 Reinjections were utilized on the second plate to obtain a total of 18 allelic ladders for this evaluation Genemapper ID X software was used to analyze each of the alleles for each of the ladders Eight second injections were determined to be standard conditions for the validated protocol and only ladders subjected to this injection time were analyzed in this study 70 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Conclusions All of the alleles in each of the ladders were consistent amongst all the other ladders This allows for the conclusion that the current procedure in validation is reproducib
5. This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice e Sample punches are incubated at 70 C for twenty minutes in an extraction buffer prior to amplification set up Samples are amplified directly from the BSD punches without extraction Sample Quantitation e The methodology was validated using a 1 2mm punch and a 25ul sample amplification volume Input DNA is managed by the sample punch size without a quantitation step Sample Amplification e Plates full of sample punches from a BSD puncher are set up for PCR AB Identifiler Plus on the Qiagen QIAgility liquid handler figure 3 Positive and negative controls are a J ai es also added to the plate at this time This liquid handler is a aandae QIAgility pre PCR instrument only e The sample plate ready for direct sample PCR amplification is removed from the liquid handler and placed directly on the Applied Biosystems 9700 Thermal Cycler Sample plates are amplified and removed from the thermal cycler Sample Genotyping e An electronic repeat pipettor is used to add formamide LIZ 600 sizing standard to a new post amplification sample plate A multichannel pipette is used to quickly and accurately add amplified DNA product
6. Volume per Number of Final Volume per Master Mix Sample uL Reactions L PCR Reaction 1 Mi 0 ix PCR Primer Set 5 Purified Water Nanopure 10 NFW or TE Add the final volume of each reagent into a sterile microcentrifuge tube Lightly vortex the PCR master mix for a few seconds and spin briefly in a microcentrifuge 30 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Add 25 uL of PCR master mix to the negative control well s and to all sample wells For the positive control add 10ul of PCR Reaction Mix 5ul PCR Primer Set and 10ul of 0 1ng ul 9947a into the applicable well Note 9947 DNA from other sources may be used at the same volume and concentration as specified above 5 Thermal Cycling Procedure A B 6 Definitions A 7 Equipment Seal the 96 well plate with an adhesive plate cover Carry the plate to the amplification room Place the plate into the 9700 GeneAmp PCR System pushing them down completely into the heat block Cover the plate with a foam compression pad to prevent evaporation Turn on the thermal cycler and select the appropriate file to initiate the cycling parameters Cycling Profile 95 C for 11 minutes then 94 C for 2
7. Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice We researched existing DNA analysis methodologies that were automated and provided sufficient throughput to address the existing and projected needs of the WSCL Each step of the automated procedure is outlined below Collection of DNA specimens e Per Wyoming State Statute all felony level convicted offenders are required to submit a DNA sample to the state Figure 1 Bode Buccal Collector database maintained at the Wyoming State Crime Laboratory e Collection is accomplished with Bode buccal collectors figure 1 Agencies from around the state submit the collectors to the WSCL where they are processed onto paper cassettes for storage efficiency and the donor s information is entered into the State DNA Database Sample Management e The process is managed by Database Manager an internally developed Excel VBA based information management system The system automates the following processes e Creating a sample group of up to ninety samples from the sample queue plus the organization of controls and ladders which are processed concurrently e Creating a BSD export file which automatically controls sample punching through the use of a barcode system This document is a
8. Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice manufacturer protocols at 28 cycles prepared with LIZ 600 v2 formamide and genotyped on the 3500 instrument Samples demonstrating possible drop out or off scale data were filtered out prior to analysis Conclusions All samples except for two demonstrated complete concordance with previously genotyped loci Samples in which the D58818 locus needed verified for true homozygosity due to a PowerPlex kit issue during outsourcing to Bode were intentionally chosen for this validation study s concordance samples Two of the D5 homozygotes were detected as being actual heterozygotes C0800235 and C0800358 The original known data generated at Bode Technologies was profiled with the PowerPlex 1 1 and 2 1 kits thereby lacking the Amelogenin D2S1338 and D19S433 loci available in the Identifiler Plus amplification kit which were not able to be compared in most samples Seven of the samples had been rerun and had previous data for these loci which was concordant with the obtained results from the validation Therefore aside from the D5 false homozygotes which account for approximately 1 of the applicable outsourced D5 homozygote samples no unexpected disconcordance was detected Notes Two samples demonstrated disconcordant alleles when compared with the known samples at the D3S1358 locu
9. adopted methodologies and protocols developed All validation studies were performed in accordance with the FBI Quality Assurance Standards QAS for databasing laboratories The final methodology validated is the direct amplification of a 1 2 mm punch from a Bode buccal collector using the AB Identifiler Plus amplification kit with electrophoresis on an AB 3500 eight capillary genetic analyzer 10 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Database Manager Software Appendix A is the validation study for the Database Manager software program an Excel VBA based sample information management system developed in house This software manages sample analysis through all steps of the process Submitted samples are assigned a unique identifier and placed within the database queue The software creates a group of up to ninety samples from the sample queue as well as placing appropriate controls and ladders in the plate setup Database Manager 2 0 General Database Info Current Lot Numbers Databasing Functions Reviewer Functions Database Queue Group Sample Maintenance System Cor 4 Database Noteboard Queue Information smcwil H No groups available X 3500 shut down 08
10. demonstrated the best results for balance at an average of 76 9 Each dye of each sample was evaluated for balance and plotted in a chart in order to demonstrate a comparison of the two buffers The samples prepared with Applied Biosystems Prep n Go buffer consistently demonstrated more balanced results in all dyes single exception of the red dye in C1100240 The greatest improvement in balance was in the green dye channel where the AB Prep n Go set demonstrated an average 19 9 balance improvement from the Bode PunchPrep samples The Bode PunchPrep sample set demonstrated a cumulative average all dyes 19 9 balance ratio as compared to a 35 3 ratio in the AB Prep n Go sample set Intralocus peak height ratios are generally used as an indicator of profile quality as low level template concentrations and or inhibition affecting the amplification reaction can lead to stochastic effects poor peak height ratios at heterozygous loci and possibly allelic dropout Intralocus peak height ratios were examined from the data obtained in both buffer sets and compared to determine if the end result electropherograms demonstrated similar or improved quality with the new AB Prep n Go buffer Results of this peak height ratio PHR study suggest significant PHR improvement with the AB Prep n Go buffer sample set Though 96 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department
11. preparation for genotyping on the 3500 The LIZ v2 0 size standard is removed from the refrigerator and mixed in proportion with the formamide to create the formamide LIZ master mix This master mix is applied to all applicable wells in a new 96 well plate in the correct volumes manufacturer recommended Amplicons from the respective plate are removed from the thermal cycler uncovered and pipetted into the formamide master mix plate with an 8 channel pipette The amplicon formamide size standard plate is covered with a 3500 septa and denatured for a few minutes followed by an ice block cooling for a few minutes The denatured plate is installed in the 3500 the 3500 analyzer allows two plates to be installed and the applicable protocols are initiated Standard injection time on the 3500 is 8 seconds though the validation supports the use of increased and decreased time injections Import files that contain sample well positions sample names and desired protocols are able to be created and imported to the 3500 software 18 2 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Analysis GeneMapper ID X software has been validated for use with data analysis at the WSCL In the course o
12. to complete the stage setup The BSD will prompt for the user to scan the first sample Scan the barcode of the first card on the plate If the correct card is scanned the BSD will prepare the punch head for sampling If the incorrect card is scanned the BSD software will return a message stating that the required card does not match the scanned card Place the desired sample under the punch head laser beam aiming for the desired punch location with the laser s location The BSD will automatically punch after the user defined delay on the BSD 25 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Xi Xil Continue punching samples 8 samples will be punched prior to the two cleaning punches until the BSD moves to the cleaning well Using the designated cleaning punch substrate punch the two cleaning punches and then continue punching samples gt Cleaning substrates may be intentionally made a different color in order that stray punches may be immediately defined as sample or cleaning punches This may allow a user to determine the source of a stray punch more easily When all desired samples have been properly punched into the 96 well plate select the appropriate option on the BS
13. 2 31 2 32 2 FGA 215 349 33 2 42 2 43 2 PET 44 2 45 2 46 2 47 2 48 2 50 2 51 2 20 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Protocol 2 Method QC measures Special QC Measures 1 Scope It is imperative that proper control samples be included when evidence samples are extracted quantified amplified and typed through electrophoresis The typing results obtained from these control samples are essential for the interpretation of STR and Amelogenin typing results from evidentiary and database samples Controls used in the WSCL Biology Unit are described below 2 Procedure A Extraction Controls Reagent Blank Reagent blank controls associated with each extraction set being analyzed must be gt Extracted concurrently gt Amplified using the same primers instrument model and concentration conditions as required by the sample s containing the least amount of DNA gt Typed using the same instrument model injection conditions and most sensitive volume conditions of the extraction set Amplification Controls Negative and Positive Amplification Controls Shall be concurrently amplified at all loci and with the same primer sets as their
14. 59 01 The 3500 will attempt three spectral calibration injections before failing b If the calibration passes Accept Results c Spectral calibration data are evaluated by the following criteria i Order of the peaks in the spectral profile blue green yellow red orange for G5 ii Order of the peaks in the raw data profile orange red yellow green blue for G5 iii Extraneous peaks in the raw data profile iv Peak morphology in the spectral profile 3 Setting up an Instrument Run A Prepare the instrument as outlined in section 5 2 B Pre heat the oven to 60 C Start Pre Heat button on main dashboard 37 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Applied Biosystems recommends pre heating the oven for at least 30 minutes before a run is started if the instrument is cold Pre heating mitigates first run migration effects Remove an aliquot of formamide from the freezer to thaw Create a plate record Import a plate record gt gt Click Create New Plate from the main dashboard view Click the down arrow next to the New Plate menu button Select New Plate from a Standard Format File in the drop down menu Select
15. C1101163 C1101170 C1101178 C1101186 C1101194 C1101202 C1101209 c1101217 C1101225 9947a Sample Punching Preparation d 00018427 00018436 00018444 00018451 00018459 00018467 00018475 00018483 00018490 00018498 00018506 BSD 600 Duet 2 C1101148 C1101156 RB1 57 C1101171 C1101179 C1101187 C1101195 C1101203 C1101210 C1101218 C1101226 AmpNeg Prep Buffer Lot 010413 12 29 2011 00018428 00018437 00018452 00018460 00018468 00018476 00018484 00018491 00018499 00018507 Heat Block 5 C1101081 C1101149 C1101157 C1101164 C1101172 C1101180 C1101188 C1101196 C1101204 C1101211 C1101219 C1101227 Heat Block Temp ri 00018361 00018429 00018438 00018445 00018453 00018461 00018469 00018477 ooo18485 00018492 00018500 00018508 C1101082 C1101150 C1101158 C1101165 C1101173 C1101181 C1101189 C1101197 C1101205 C1101212 C1101220 C1101228 PCR Setup Amplification 00018362 00018430 00018439 00018446 00018454 00018462 00018470 00018478 ooo1sass 00018493 00012501 00018509 ID Plus Lot 091612 C1101087 C1101151 C1101159 C1101166 C1101174 c1101182 C1101190 C1101198 RB2 57 C1101213 C1101221 C1101229 Water Lot 011113 00018367 00018431 00018440 00018447 00018455 00018463 00018471 00018479 00018494 00012502 00018510 12 29 2011 QlAgility 1 C11
16. Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice there were several independent instances in which a decreased PHR was obtained with the AB buffer the average sample s locus demonstrated an improvement especially at the larger fragment loci Three loci D21 CSF and D3 showed a decrease in PHR with the AB Prep n Go buffer though not a significant drop D3 was the greatest drop from 87 3 on the Bode buffer samples to 85 0 on the AB buffer samples One sample in the AB buffer set C1100240 demonstrated a very large improvement 28 0 to 77 8 a 178 increase in peak height ratio On review of the electropherograms the D18S51 genotype of 16 22 gave peak heights of 5585 4343 respectively on the AB buffer set and 2546 712 on the Bode buffer set As this sample and the rest of the data indicate an overall improvement of PHR the AB buffer on average appears to yield a more quality PHR The final analysis of the data was a comparison of combined peak heights at each locus of each sample whereupon the buffer sets were contrasted to determine amplitude differences On average combined peak heights were significantly improved in the AB buffer sample set Further examination of the results by fragment size shows that the small fragment loci are less affected than the medium and larger loci As the results obtained in routine datab
17. U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Executive Summary The Wyoming State Crime Laboratory WSCL is statutorily obligated to perform offender sample profiling through Wyoming State Statutes W S 7 19 401 through 7 19 406 which call for the establishment and administration of a Wyoming State DNA Database and allows for participation in NDIS Historically the WSCL complied with this obligation through outsourcing samples to private DNA laboratories with the help of funding through the NIJ as there was no equipment or laboratory space at the WSCL dedicated for CODIS sample profiling We sought a long term solution to this issue by the establishment of an automated fully functioning CODIS dedicated laboratory The Wyoming State Legislature funded the construction of a new state laboratory building complex in Cheyenne Wyoming Included in the building complex is over one thousand square feet of space dedicated to CODIS laboratory and CODIS administrative functions The NIJ funding through this grant allowed for the purchase of the laboratory testing equipment necessary to achieve our goal which was a fully functioning laboratory dedicated to CODIS convicted offender DNA profiling This document is a research report submitted to the U S
18. associated forensic samples All samples typed shall also have the corresponding amplification controls typed Positive Amplification Controls 9947a is a positive control for STRs and amelogenin to evaluate the performance of amplification and electrophoresis When the control specimen 9947a is amplified the STR loci must solely exhibit the correct genotype Additionally 9947a is the control for Amelogenin and must exhibit a single band at the position corresponding with the size ladder band representative of the peak from the X chromosome Negative Amplification Control A Negative Control must be included with each set of amplifications The negative control contains all components required for the amplification of DNA except that no DNA is added A volume of nuclease free water 21 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice C D 3 Definitions A D 4 Equipment A equal to that of the sample amplified is placed in the negative control in lieu of a DNA solution This control is processed through the amplification and electrophoretic typing procedures Quantitation Where quantitation is used quantitation standards shall be used The DNA procedures shall be ch
19. by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice 2 Barcoding of samples to use on the BSD sample puncher As samples are generally sequential any breach of the sequence would be immediately detected in the BSD software As sample lists are given to the BSD prior to the punch run samples out of order would be detected right away Similarly if any samples in the sequence were previously removed or expunged from the biographical database and the physical sample was not destroyed the BSD would alert the user to the absence of the record from the list 3 QIAgility PCR setup will standardize sample setup and will perform the same protocol in replicate much more consistently than a human analyst In addition the analyst has free time to setup downstream instrumentation or clean up BSD related workspaces while the PCR setup is in progress 4 3500 input files generated from the same list as the BSD input files will ensure that samples will fall parallel in the plate setup throughout the procedure This input file will save analyst time avoid transcription errors and keep the virtual sample setup integrity intact Validation Specific Equipment Information 1 BSD Robot 1 Model 600 Duet S N 10071 2 BSD Robot 2 Model 600 Duet S N 10079 3 QIAgility 1 S N 00306 4 9700 Thermal Cycler 5 S N 805S0210800 5 97
20. decreased poor balance The 0 0125ng ul group had a large increase in peak height imbalance and partial and or total dropout at multiple loci Approximately 15 of the alleles in this sample group demonstrated dropout The 0 00625ng ul through 0 001563ng ul groups showed significant dropout with the lower concentrations in this range demonstrating nearly complete dropout only 3 of the 246 expected alleles were detected at the 0 001563ng ul level Stochastic Effect Results All samples from the sensitivity study subjected to the standard 8 second injection time on the 3500 analyzer were investigated for false homozygote peaks Known profiles for each of the four samples used in the study were compared against the obtained 64 sensitivity samples 4 samples x 7 dilutions x 2 injections each All truly heterozygote loci demonstrating a single peak were flagged for being a false homozygote peak From that pool of false homozygotes each obtained allele was compared to determine the highest false homozygote peak After all peaks were reviewed the maximum was a 427 RFU 27 allele sample OOF0069 peak at D21811 with Figure 16 Highest false homozygote observed in study a partner 32 2 allele that did not break the 150 RFU 0 0125n ul detection threshold see figure 16 The next highest false homozygote detected in this study was a 385 RFU peak at D7S820 In summary whenever a single peak was detected gt 427 RFU it was a true
21. for participation in the National DNA Index System NDIS Historically in Wyoming offender sample processing was being performed only by outsourcing offender samples to private laboratories using funding from the National Institute of Justice NIJ There was no equipment or laboratory space at the Wyoming State Crime Laboratory WSCL dedicated for CODIS sample profiling We sought to address this mission critical need by the establishment of an automated fully functioning CODIS dedicated laboratory The NIJ funding through this grant allowed for the purchase of the laboratory test equipment necessary to achieve this goal The Wyoming State Legislature funded the construction of a state laboratory building complex in Cheyenne Wyoming which was completed in November 2010 Included in the building complex is over one thousand square feet of space dedicated to CODIS laboratory and CODIS administrative functions The goals and objectives of this project were to establish and increase the capacity of the WSCL CODIS Unit in order to meet This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice the current and anticipated critical need for a highly automated fully functioning CODIS Laboratory by the procurement
22. for that sample at that locus may be designated as inconclusive INC Samples demonstrating inconclusive alleles at current NDIS core loci are not eligible for database entry No result For those samples where there are no peaks at a particular locus this locus is designated with an NR or negative no result Controls and Standards i Ladders Alleles must be correctly genotyped and the peak height must be 150 RFU or greater Resolution should be sufficient to distinguish a single base difference At least one 1 ladder per panel per run must type correctly See Biology Exhibit 27 02 ID Plus User Guide for Identifiler Plus ladder alleles ii Internal standard Fragments must be labeled and sized correctly in order to report the corresponding sample The internal standard fragments must bracket the alleles being sized iii Reagent blank No typed alleles present If the reagent blank exhibits a DNA profile at a specific locus or loci any sample s concurrently extracted with this control are considered inconclusive iv Amplification Positive Control Typed alleles must match expected alleles If the expected alleles are not detected in the positive control then any sample s concurrently typed with this control are considered inconclusive Other appropriate Human DNA Controls may also be used Reference the DNA CE Manual for other possible factors which may influence the interpretation of the positive quality c
23. height ranged from 5 7 blue to 22 9 RFU red The standard deviation of the dyes ranged from 4 8 blue to 9 1 RFU yellow The maximum peak height observed was at 169 RFU in the yellow dye channel Many of the higher background peaks observed were not in a locus range but were to the left of the smaller loci which was the case with the 169 RFU 78 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice maximum peak in the yellow channel see figure 20 Combining all dyes the average background peak was 13 6 RFU with a 6 9 RFU standard deviation 3500 Identifiler Plus Background Averages by Dye 8s Average RFU Yellow All Bue ereen vellow Red C _78 __97 160 22 109 std Dev _ 4 853805 6 323156 9 093158 7 165216 6 886898 Figure 19 Background study results Figure 20 Largest background peak highlighted on left detected in study was outside of loci ranges in the yellow dye 169 RFU Conclusions The data based on these results gave an instrument limit of detection LOD mean 3Sd of 34 2 RFU and a limit of quantitation LOQ mean 10Sd of 82 4 RFU Taking into account the LOQ a more conservative 150 79 This document is a research
24. homozygote peak 75 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Sensitivity Conclusions Samples amplified with a total amount of DNA between 0 2ng ul and 0 05ng ul demonstrated full profiles when injected under standard conditions and analyzed with a 150 RFU detection threshold Though samples amplified at 0 2ng ul exhibited mostly acceptable or recoverable with a decreased injection time profiles it should be noted that the study was intentionally conducted with highly heterozygous samples which require more DNA to become off scale Samples demonstrating more realistic variations of homozygote loci may be extremely off scale when amplified from 0 2ng ul template DNA levels Samples from 0 025ng ul and lower in concentration demonstrated dropout similar results should be analyzed with caution if they are encountered in database or casework applications The optimal template DNA concentration for the Identifiler Plus on the 3500 genetic analyzer under the conditions utilized in this validation procedure is between 0 05ng ul and 0 1ng ul Based on this optimum the best template DNA concentration for amplification of this chemistry is 0 075ng ul Stochastic Effect Conclusions Due to the highes
25. ii Pipette tips iii Microcentrifuge iv Vortex v Pipettes vi lonization Bar Materials Note Refer to Biology Chemistry Manual for specific information i Bode PunchPrep solution non FTA samples only ii AB Prep N Go non FTA samples only iii Purified water humidification system iv 96 well amplification plates Reagents N A Calculations N A Uncertainty of Measurement N A Limitations A 10 Safety A B 6 7 8 Acceptance Criteria N A 9 Small paper punches e g 1 2mm punches from the BSD are subject to increased effects of static charges and air currents due to their small mass Users should closely monitor all sample runs to ensure the plate s integrity Safety precautions shall follow the WSCL Safety Manual Turn off the BSD prior to working or cleaning inside the machine Multiple fast moving axes are available to the instrument which may cause injury to users with body parts inside the active instrument Though the radiation from the ionization bar has been deemed safe alpha emission from Polonium 210 for general use it should not be ingested or subjected to prolonged contact exposure to skin and or other body surfaces The Amstat 2U500 ionization bar produces ionization 27 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the
26. in obtaining an acceptable DNA profile will be transferred to the casework system when necessary The casework process may include extraction purification and quantitation steps in attempt to give the sample the best chances of yielding a DNA profile Samples profiled in the casework path may differ in amplification chemistry as long as the current NDIS definition of core loci at the applicable databasing indexes is satisfied by the casework kit A Substrate Sampling Pre PCR Treatment Bode buccal collectors are sampled via a 1 2mm punch on a semi automated BSD 600 Duet sample puncher Direct amplification methods typically require samples from an FTA paper which are theoretically lysed cells with the DNA bound to the paper As the buccal collectors used at the WSCL are only a filter paper Bode purchases the filter paper from Whatman as per correspondence 16 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice with GE Whatman representative Betsy Moran February 2011 an additional reagent is required to lyse the cells prior to PCR setup 2ul of a product developed from Bode Technologies Bode PunchPrep or 2ul of a product developed from Applied Biosystems AB Prep N Go are used per well wi
27. message on the computer regarding sample number expectations In the case of a correct sample scan the BSD will position the plate below the sample punch chute and activate the punch head The database analyst will position the buccal collector sample under the punch spot a red laser dot gives precise position of the area to be punched and will activate the BSD to execute the punch The punch will fall through the chute and into the desired well Eight samples will be allowed to be punched followed by a two punch cleaning punch which goes into a large trash can well beside the plate In order from the beginning the import file for the BSD is loaded onto the computer applicable wells in the sample plate are pipetted 2ul of Bode PunchPrep the sample plate is irradiated the plate is installed on the front position of the BSD robot two 96 well positions exist on the robot samples are scanned and punched into their respective wells and the plate is removed from the BSD and installed on the heat block where it is incubated at 70 C for 20 minutes At this point the sample plate is ready for PCR setup PCR Setup Though manual preparation and dispensing of master mix into the sample plate is allowed a protocol on the QIAgility liquid handler has been 66 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of
28. not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice AB 3500 ID Plus Validation Stutter Study Summary Introduction Using the Identifiler Plus PCR kit approximately 110 of the concordance sample electropherograms were analyzed in GeneMapper ID X to determine maximum observed stutter levels for each locus Obtained values were compared against manufacturer published maximum stutter data wherein the greater of the two values was adopted for use with the procedure Methods Electrooherograms were generated according to the validation procedure for direct punch amplifications Data used in this background study is almost the exact data set used in the concordance study Samples were analyzed at a decreased 60 RFU threshold in order to visualize more stutter below the 150 RFU detection threshold The ID Plus panel set was modified to filter 0 stutter at all loci thereby detecting all peaks Results Average observed N 4 stutter percentages ranged from 8 22 D18S51 to 2 59 THO1 Maximum stutter percentages ranged from 14 96 D18S51 to 6 18 TPOX Other types of stutter were recorded including N 4 N 8 and N 4 N 8 N 4 N 4 and N 4 N 8 combination stutter peaks In general the N 4 and N 8 stutter peak data should serve as evidence to explain these peaks when they are encountered in routine d
29. or otherwise too much input DNA Pink failed low quantity or otherwise too little input DNA Yellow Tri allele or rare variant rerun Uncolored samples may be new samples or rerun samples with an appropriate DNA input from the original run e Manages reanalysis of samples that show unsatisfactory results or require additional analysis such as tri alleles e The group and sample maintenance interface provides for a searching by status e g completed active or failed or group sample number see figure 11 Group or sample targets can be modified in priority name or phase and failed samples can be tracked and completed as applicable e Auditing functions allow access to upper level users for viewing all date time information associated with any sample whether failed active or completed Database Manager 2 0 General Database Info Current Lot Numbers Databasing Functions Reviewer Functions Database Queue Sroup Sample Maintenance system Cor Group Info Maintenance Sample Info Maintenance Manual Sample Processing Goo0s7 X Choose a sample X Status Phase 0 Members 9 samples v Generate List of Manual Samples Complete Failed Samples New Sample in Group Modify Group Phase Logged in user smewil Figure 11 Group and sample management interface 15 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or point
30. plate When the ionization bar was approximately 1 2 inches from the plate punches that adhered to the sides of their respective wells immediately dropped to the bottom of their wells After witnessing this all plates and the lower BSD components were subjected to ionization prior to a sample punching run This method effectively increased the success rate to approximately 99 though the user is still recommended to keep a close watch on the punching in order to discover problems as they occur 4 Cleaning Punch Color Coding Possibly the most simple solution suggested was a different color for cleaning punches In the course of the validation any stray cleaning punches falling in sample wells were easily identifiable and removed when detected After the sample plate ionization 91 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice was employed the carryover of cleaning punches was not as frequent though the color coding was still a great visual aid QIAgility Setup Optimization The QIAgility liquid handler was used as the main tool in the PCR setup steps of the direct amplification protocol Multi dispense pipetting on the QIAgility is much less pipette tip intensive and is far faster than
31. precision of the procedure using an appropriate control s Precision Study Reproducibility Study 63 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice 3 3 Match criteria For procedures that entail separation of DNA molecules based on size precision or sizing must be determined by repetitive analyses of appropriate samples to establish criteria for matching or allele designation Precision Study 3 4 Sensitivity and stochastic studies The laboratory must conduct studies that ensure the reliability and integrity of results For PCR based assays studies must address stochastic effects and sensitivity levels Sensitivity Study Background Study 3 5 Contamination The laboratory must demonstrate that its procedures minimize contamination that would compromise the integrity of the results A laboratory should employ appropriate controls and implement quality practices to assess contamination and demonstrate that its procedure minimizes contamination Contamination Study 3 6 Qualifying Test The method must be tested using a qualifying test This may be accomplished though the use of proficiency test samples or types of samples that mimic those that the laboratory routinely analyzes This qualifyi
32. report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice RFU threshold will be adopted to filter out the majority of background peaks Though some background peaks e g peak outlined in figure 20 exceed the 150 RFU threshold the majority of high peaks are outside of expected loci ranges and would not be considered in actual casework or databasing applications Notes The background of the 3500 instrument is significantly elevated as compared to the 3130 instrument In a comparison of this study ID Plus ona 3500 to an Identifiler 3130 background validation study the trends in background dye averages and standard deviations are similar though the 3500 data averages are at notably higher RFU values see figure 21 Conversations with the representatives from Applied Biosystems during training events on the 3500 prior to the validation study had included information about the new detection system solid state laser in the 3500 which ultimately yield a considerably higher detection and stochastic thresholds Average 3500 and 3130 Background Levels Yellow Figure 21 Background level comparison between 3500 and 3130 instruments 80 This document is a research report submitted to the U S Department of Justice This report has
33. the author s and do not necessarily reflect the official position or policies of the U S Department of Justice validated along with the study Through the preliminary stages of the validation the optimal PCR mix was found to be the manufacturer recommended 10ul of PCR Reaction Mix with 5ul of PCR Primer mix and 10ul of water with the 1 2mm sample punch The 10ul of water was added in place of 10ul of DNA extract as the sample punch in the 10ul water was theorized to be analogous to an extract and found to be acceptable through this validation study Prepared sample plates are installed on the QIAgility deck with applicable consumables and Identifiler Plus PCR reagents The protocol is executed which will create and dispense the master mix in all applicable wells Control 9947a DNA is added to the respective positive control well and the water used in the protocol is sampled to create an amplification negative in the respective negative control well Approximately 7 minutes is required to complete this protocol with a full plate on the QIAgility liquid handler Upon completion of the QIAgility protocol or manual dispensing of liquid plate contents an adhesive plate cover is applied At this point sample plates are ready for amplification in the thermal cycler Amplification Covered sample plates are transferred to a 9700 thermal cycler where a compression pad is placed on top of the covered sample plate to prevent evaporation of plate conten
34. water was theorized to be analogous to an extract and found to be acceptable through this validation study Prepared sample plates are installed on the QIAgility deck with applicable consumables and Identifiler Plus PCR reagents The protocol is executed which will create and dispense the master mix in all applicable wells Control 9947a DNA is added to the respective positive control well and the water used in the protocol is sampled to create an amplification negative in the respective negative control well Approximately 7 minutes is required to complete this protocol with a full plate on the QIAgility liquid handler Upon completion of the QIAgility protocol or manual dispensing of liquid plate contents an adhesive plate cover is applied At this point sample plates are ready for amplification in the thermal cycler C Amplification Covered sample plates are transferred to a 9700 thermal cycler where a compression pad is placed on top of the covered sample plate to prevent evaporation of plate contents All sample wells contain a 1 2mm punch and Identifiler Plus master mix Through preliminary validation plates the optimal number of cycles was found to be 28 which is the cycle number on the Identifiler Plus Database protocol on all applicable thermal cyclers The sample plate is installed on the thermal cycler and the protocol is initiated which takes approximately 3 hours D Genotyping on 3500 Analyzer Frozen formamide is thawed in
35. 0 seconds 59 C for 3 minutes for 28 cycles then 60 C for 25 minutes then 4 C hold Note The entire cycle takes approximately 3 hours DNA type or DNA profile is the genetic constitution of an individual at defined locations loci in the DNA A DNA type derived from nuclear DNA using STR technology typically consists of one or two alleles at several loci DNA technology is the term used to describe the type of forensic DNA analysis performed in the laboratory such as RFLP STR YSTR or mitochondrial DNA Test kit is a pre assembled set of reagents that allow a user to conduct a specific DNA extraction quantitation or amplification Materials and Reagents 31 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice A Equipment Note Refer to Biology Equipment Manual for specific information Bio safety hood QIAgility liquid handler Microcentrifuge iv 9700 GeneAmp PCR System v Pipettes vi Vortex B Materials Note Refer to Biology Chemistry Manual for specific information 9947 control DNA TE Buffer Nuclease free water iv Microcentrifuge tubes v 96 well amplification plates C Reagents Note Refer to Biology Chemistry Manual for specific informatio
36. 00 9 13 11 9 13 2011 ee il QC testing ID lot 080812 today 9 6 2011 F il New ID Plus kits need QC Lot 080812 9 2 2011 Active Samples 91 samples available v il Taking GO0042 G00043 today QC test this morning 8 29 2011 il New ID Plus kits 3 should be here tomorrow 8 9 2011 il Only one full plate of ID Plus reagent left right now 7 25 2011 il G00036 preliminary review looks good send it to TR phase 7 13 2011 Total samples 91 created GO0036 7 7 2011 smcwil BG you are ready to punch and create a group 7 6 2011 i smewil BG make a new group from queue and start phase 1 7 6 2011 Grant Information smcwil Sample queue synchronized for DBM2 today 6 27 2011 2009 Grant gt C0800959 to C1000344 2010 Grant gt C1000345 to C1100726 2011 Grant gt C1100727 to Unassigned samples 91 Logged in user smewil Figure 6 Main interface of Database Manager The functions in Database Manager allow for sequential movement of databasing samples throughout the process and automatically provide the following functionality e Creating a BSD export file which controls sample punching through the use of a barcode system on the BSD 600 see figure 8 Five distinct stages in the 11 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily
37. 00 Thermal Cycler 6 S N 80580202494 6 3500 Genetic Analyzer S N 22118 131 93 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Capillary Array Usage Notes Though the manufacturer recommends 160 maximum injections per capillary array for 3500 8 capillary arrays reference AB Website the application specialist for the WSCL April Orbison May 2011 has stated that just as the 3130 arrays last for more injections with frequent injections and low stagnation the 3500 arrays will last for more injections if they are used frequently As with the 3130 s the quality of the peaks in the electrooherograms should guide the users to determine if the capillary array needs changed out In addition to the capillary array injection recommendation 3500 capillary arrays now have an expiration date which per the WSCL AB representative is to discourage stock piling of arrays as they deteriorate in quality over the course of a couple years The expiration date is not a hard stop on the 3500 hard stops require the user to modify a setting prior to continuing and can be ignored though the expiration date is logged in the resultant hid electrooherogram file See table below for detail about reagents and
38. 01152 C1101160 C1101167 C1101175 C1101183 C1101191 C1101199 C1101206 C1101214 C1101222 C1101230 Thermal Cycler 5 00018432 00018441 00018448 00018456 00018464 00018472 00018480 00018487 00018495 00018503 00018511 C1101145 C1101153 C1101161 C1101168 C1101176 C1101184 C1101192 C1101200 C1101207 C1101215 C1101223 Ladder Genotyping Data Collection Date 00018425 00018434 00018442 00018449 00018457 00018465 00018473 00018481 00018488 00018496 00018504 Formamide Lot 110312 12 29 2011 Ezeren ozz 898ul formamide 52 8ul LIZ 9ul well POP4 Lot 041212 Anode Buffer Lot 040812 Cathode Buffer Lot 040112 3500 Analyzer 1 12 29 2011 All kit components are the same lot number unless otherwise specified 3A Database Sample Worksheet Revision 1 1 Issue Date 09 27 2011 Authorized By Bill Gartside Working Copy when Outside the LQAD Page 1 of 1 14 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Figure 10 Database group record sheet example Rerun samples may have color coded wells Green failed off scale
39. D Plus Validation Increased Injection Time Summary Summary All sensitivity stochastic study samples were subjected to an increased 15 second injection time N 32 In all cases excluding dropout events in the 8 second injection samples allele calls obtained from the increased injection times were concordant with the 8 second standard injections A brief comparison of the 8 and 15 second electropherograms demonstrated an approximate average peak height gain of 250 to 400 when performing a 15 second injection after the original 8 second injection This study provides support for a decreased time injection when necessary e g off scale data Notes Increased injection times may be applied when dropout has occurred at one or more loci As in the previously validated Identifiler kit homozygote alleles below the stochastic threshold 450 RFU cannot be salvaged with an increased injection time These special injections should be applied in the attempt of raising one or both peaks of heterozygote loci above the detection limit Caution should be exercised when applying the increased injection times and the applicable reagent blank on the 96 well plate should be subjected to the same conditions of the sample injection 88 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily ref
40. D form e g All Spots Present and select Yes to exit the program If using non FTA samples the Bode PunchPrep or AB Prep N Go must be incubated gt Place the 96 well plate on the heat block for 20 minutes at 70 C With caution to not drop the sample plate transfer the plate to the appropriate location in the PCR setup area in preparation for direct amplification 3 Maintenance Procedure A General Maintenance performed after each run Clear visible debris from punch platform on BSD and inside BSD If cleaning inside the BSD ensure the instrument is turned off for your safety Organize and clean bench top spaces around the BSD instrument If necessary clean with a diluted bleach solution followed by an ethanol wipe gt Never use ethanol on the plastic components of the BSD sample puncher Check that the cleaning punch well is not full and that the cleaning punch substrate s have adequate material left for additional runs 4 Definitions N A 5 Equipment Materials and Reagents 26 This document is a research report submitted to the U S Department of Justice This report has not C been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Equipment Note Refer to Biology Equipment Manual for specific information i BSD 600 Duet sample puncher
41. Figure 8 Databasing interface Controls on this page allow for sample progression 13 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Database Manager 2 0 General Database Info Current Lot Numbers Databasing Functions Reviewer Functions Database Queue Group Sample Maintenance Review Tools iew Groups Group G00057 Tech Review Group Open Paradigm 3 Members 90 Group Notes C1101081 a Triallele on sample C1101252 MY 11 1 C1101082 on sample C1101275 C1101087 Open Active C1101106 off scale sample C1101266 resubmitted Folder C1101145 to queue C1101146 C1101147 C1101148 Print List of C1101149 Group Members C1101150 C1101151 Logged in user jbramm Datanse Manager v20 Figure 9 Reviewer interface Allows for organized access to review groups and associated information Sample Group Information Database Plate Setup 1 2 3 4 5 6 7 8 9 10 1 12 C1101146 C1101154 C1101162 C1101169 C1101177 C1101185 C1101193 C1101201 C1101208 c1101216 C1101224 Ladder j T Lab Biology Casework Images CODIS 00018426 00018435 00018443 00018450 00018458 00018466 00018474 00018482 00018489 00018497 00018505 C1101147 C1101155
42. IS Unit in order to meet the current and anticipated critical need for a fully functioning CODIS Laboratory by the procurement of dedicated CODIS Unit laboratory equipment The equipment has been procured and we feel the goals and objectives have been met References and Dissemination of Project Findings A short paper about this process which highlights the differences in results when using various buffers has been submitted to Applied Biosystems for possible inclusion into their trade publication Forensic Laboratories interested in acquiring the database manager software developed at WSCL should please contact the authors There is no charge for the software which may be made available by request with certain limitations 56 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Appendix A Database Manager v2 0 Validation Check Software Introduction The Database Manager software has been an excellent tool in sample and group organization as well as worksheet automation throughout the course of databasing CODIS samples in house Due to a grant allowing the Wyoming State Crime Laboratory to develop a databasing system a direct amplification procedure was developed and validated with t
43. Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Fill a 2 0ml Qiagen sample tube with purified water or TE and install it in position C of the reagent plate Place the 9947a positive control tube in position H of the reagent plate Load the 96 well plate containing all punched samples into position C2 lower right hand corner of QIAgility deck Initialize the PCR setup run by pressing the green play button on the toolbar Acknowledge any applicable maintenance reminders and continue to begin the run Save the post run report in the Run Archive folder Put away unused reagents and clean any necessary work surfaces Restock consumables that are empty on the QIAgility Shut down the QIAgility instrument and then the computer Open the hood containing the adhesive plate covers in preparation for sealing the plate 4 Procedure Manual PCR Setup A Determine the number of reactions to be set up This should include reagent blanks and positive and negative amplification controls 1 or 2 reactions may be added to this number to ensure an adequate amount of PCR Master Mix For samples and negative controls calculate the required amount of each component of the PCR master mix Table below Multiply the volume uL per sample by the total number of reactions to obtain the final volume uL
44. State Crime Laboratory AB 3500 ID Plus Validation Introduction A new Applied Biosystems 3500 genetic analyzer was purchased in late 2010 with assistance from a NIJ grant This 3500 instrument will function as a dedicated database instrument in a direct amplification system Two BSD 600 Duet sample punchers provide an organized solution to placing individual 1 2mm sized sample punches into a 96 well plate format the plates with their respective punches are added to a QIAgility liquid handler that creates and adds an Identifiler Plus master mix to the appropriate sample and control wells in the plate the plates from the QIAgility are sealed and placed on the thermal cycler for amplification the amplicons are added with a multi channel pipette to a formamide master mix and genotyped on the 3500 genetic analyzer All data analyses from the genetic analyzer will be performed in the GeneMapper ID X v1 2 software The majority of this validation study will be directed toward the amplification chemistry on the genetic analyzer though the supporting instrumentation and method development will be discussed where applicable This validation will primarily serve to establish background stutter sensitivity stochastic limits and thresholds as well as verifying the precision reproducibility concordance obtained peak height ratios and effects of modified injection times These values will be obtained through data generated on the 3500 instrument The fol
45. The author s shown below used Federal funds provided by the U S Department of Justice and prepared the following final report Document Title Filling a Critical Need by Establishing a Fully Functioning CODIS Dedicated Laboratory Author Bill Gartside Scott McWilliams Document No 238909 Date Received June 2012 Award Number 2009 DN BX K249 This report has not been published by the U S Department of Justice To provide better customer service NCJRS has made this Federally funded grant final report available electronically in addition to traditional paper copies Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Filling a Critical Need by Establishing a Fully Functioning CODIS Dedicated Laboratory within the Wyoming State Crime Laboratory Final Technical Report FY 2009 Forensic DNA Unit Efficiency Improvement Program Award Number 2009 DN BX K249 Author s Bill Gartside Scott McWilliams l Abstract Wyoming State Statutes require the establishment and administration of a Wyoming State DNA Database and allows
46. air through a damp wet sponge system for humidification prior to entering the punch head on the BSD robot Upon investigation it was learned that the airflow from the humidification system travels down from the punch surface through the punch chute and dissipates As some punches at this stage were witnessed to have bounced out of their respective wells the humidified 90 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice airflow was reduced and the numbers of successful punches were increased 3 Sample Plate lonization Though the success rate of the sample punching after application of solutions 1 and 2 above was acceptable static electricity was still an issue causing samples to stick to both the BSD punch detector and the tops of the wells plate at times Different attempts to combat the static electricity were employed from dipping the bottom of the plates in water prior to punching to violently tapping the plates on the counter Creative discussions about grounding the BSD to the user electrically were brought up though eventually an atomic ionization device was purchased through Amstat Industries part number 2U500 The first ionization with this device was attempted on a pre punched
47. amide amplicon LIZ 600 v2 preparation The plates were created on different days 03 28 2011 and 04 05 2011 Reinjections were utilized on the second plate to obtain a total of 18 allelic ladders for this evaluation Genemapper ID X software was used to analyze each of the alleles and their respective sizing for each of the ladders Eight second injections were determined to be standard conditions for the validated protocol and only ladders subjected to this injection time were analyzed in this study Conclusions 3x the standard deviation was calculated for each allelic bin on the Identifiler Plus ladder When all allelic 3xSd values were averaged their respective total locus 3xSd values the range was from 0 090 D3S1358 base pairs to 0 180 base pairs D8S1179 These results support the conclusion that the procedure used in the course of this validation is capable of resolving differences in length by one base pair and that the 0 5 base pair bin sets used by the GeneMapper ID software is appropriate for accurate allele calls 69 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Sizing Variance 3xSd per Locus Precision Study E Average a 7 7 co E Max
48. ase processes have demonstrated ample small fragment peak height and lower than desired large fragment peak heights the increases in peak heights at these medium and large fragment loci shows evidence for improved overall electropherogram quality by using the AB buffer Decreased combined peak heights were witnessed on multiple loci of a single sample C1100553 with the 97 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice AB buffer set Upon examination of the respective electropherograms both samples appear to be good quality DNA profiles with ample peak height and decent interlocus balance however the AB buffer sample demonstrated an approximate 9 0 gain in peak height ratio quality The D2S1338 locus demonstrated the greatest average amplitude gain 503 in the AB buffer sample set All locus averages indicated amplitude gains favoring the AB buffer sample set over the Bode buffer sample set Though not part of the core study it should be mentioned that all samples profiled with AB Prep n Go demonstrated results concordant with the previously validated method incorporating the Bode PunchPrep buffer Conclusions The AB Prep n Go buffer sample set treated identically to the Bode PunchPrep sa
49. atabasing and casework applications The combination stutter peaks are generally exhibit an additive effect between the two stutter peaks The most combination effect appears when two alleles at a given locus are separated by 2 repeat units thereby giving an N 4 N 4 peak The average of the N 4 N 4 combination peaks was higher than 81 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice the average N 4 at every respective locus tested in this study Combination stutter peaks should be considered when evaluating minor peaks above stutter percentage thresholds The highest N 4 observed was 8 42 of the parent peak vWA and the highest N 8 peak observed was at 4 15 D58818 of the parent peak Conclusions When a comparison was made between the WSCL obtained N 4 maximum stutter peaks and the manufacturer s published stutter data reference Identifiler Plus User s Manual WSCL stutter was higher at 9 of the 15 loci Therefore stutter values were adopted from both data sets using the maximum value at each locus see data on following chart table referencing the N 4 comparison Less common types of stutter e g N 8 N 4 and combination stutter peaks may be characterized for which this validation stu
50. author s and do not necessarily reflect the official position or policies of the U S Department of Justice approximately 2 from the surface past which there is no radiation due to the absorption in the surrounding air 11 Report Writing N A 12 References and Exhibits A Exhibit 8 Manufacturer s technical manual s data centrifuges B Exhibit 9 Manufacturer s technical manual s data vortexes C Exhibit 10 Manufacturer s technical manual s data pipettes D Exhibit 60 Manufacturer s technical manual s data BSD 600 Duet 13 Record Keeping E Plate setup records will be physically or electronically archived with the applicable sample group s records and archive 14 Forms 3A Database Sample Worksheet 28 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Protocol 4 Direct Identifiler Plus amplification Direct Amplification with Identifiler Plus All PCR setup steps must be performed in a pre amplification hood or liquid handler using reagents and pipettors dedicated to this area 1 Scope The Identifiler Plus kit is a test kit containing the reagents necessary for performing genetic typing This technical SOP governs the
51. ctrophoresis These allelic ladders contain the more common alleles in the general population for each locus Using the ladders the alleles present in known and questioned DNA specimens may be determined The following table lists the Identifier Plus loci the size ranges of alleles within a particular locus the alleles present in the ladder and the fluorescent label 19 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice STR Size Range Alleles Present in Fluorescent Label Locus bp Ladder D8S1179 128 172 7 20 6 FAM 24 24 2 25 28 28 2 29 29 2 30 30 2 D21S11 189 243 31 31 2 32 32 2 6 FAM 33 33 2 34 34 2 35 35 2 36 38 D7S820 256 294 6 15 6 FAM CSF1PO 306 342 6 15 6 FAM D3S1358 114 142 12 19 VIC THO1 165 204 4 9 9 3 10 11 13 3 VIC D13S317 217 245 8 15 VIC D16S539 261 297 5 8 15 VIC D2S1338 309 361 15 28 VIC 9 12 12 2 13 13 2 D19S433 110 140 14 14 2 15 15 2 NED 16 16 2 17 17 2 vWA 157 209 11 24 NED TPOX 225 253 6 13 NED 7 9 10 10 2 11 12 D18S51 269 341 13 13 2 14 14 2 NED 15 27 Amelogenin 107 X 113 Y X Y PET D5S818 135 171 7 16 PET 17 26 26 2 27 30 30
52. dy should serve as a reference Notes A few anomalies of N 4 stutter were removed from this study due to significantly elevated stutter percentages See the associated electropherograms in the validation binder stutter study for more detail on these omitted outliers 82 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Average Identifiler Plus N 4 Stutter Per Locus 7 Led 8 E D18551 i D24511 i i paseis WA D8S1179 6 ee D5S818 DT69539 D7S826 SkBPO D13S317 4 THO1 Percent Stutter Observed 2 0 Figure 22 Stutter study results Maximum Stutter Comparison Between WSCL and Applied Biosystems Data N 4 Percent Stutter Observed Figure 23 Comparison between WSCL and Applied Biosystems observed stutter percentages AB 3500 ID Plus Validation Peak Height Ratio Study Summary Introduction 109 samples used in the concordance study were analyzed to determine peak height ratios at all heterozygous loci Samples used in this study 83 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect th
53. e Groups currently in phase 0 in a group but not further in the process are allowed to proceed to phase Phase creates a sample worksheet with controls applied a BSD import file a 3500 import file and updates the queue to show the group in phase The BSD import files successfully import on the BSD instruments and the target offender samples are correctly read by the BSD barcode reader The 3500 instrument successfully imports the plate setup file and consistently shows the proper plate sample setups These functions work as expected Phase Il Groups currently in phase are allowed to proceed to phase II Phase II applies a genotyping date to the sample worksheet and updates the queue to show the group in phase II This function works as expected Phase lll Groups currently in phase Il are allowed to proceed to phase III Phase III imports a genotyping summary file from the analyst s analyzed data out of GeneMapper ID X and creates a summary allele table The summary allele table is saved in the group record file and any reoccurring genotypes are flagged as possible contamination in an orange color The queue is updated to show the applicable group samples in phase Ill These functions work as expected Submit for Tech Review Groups currently in phase III are allowed to be locked for technical review This process updates the queue to show the group in phase IV which in turn makes the group visible to the reviewer tab when a differe
54. e interpretation of the loci do not need to be re amplified Shoulders shall be marked as such within the case notes gt A samples with peak heights near maximum threshold may exhibit incomplete non template nucleotide addition A Samples with excessive incomplete non template nucleotide addition may be re amplified with less DNA A shall be marked as such within the case notes gt Pull up the result of the matrix not fully correcting for spectral overlap of the dyes and is most often caused by an excessive amount of DNA or suboptimal matrix Pull up peaks typically size within 2 scan numbers as the true peak 46 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Spikes caused by transient fluorescent materials in the injection as well as electrical impulses Spikes can occur in one two three four or five colors and will disappear upon re injection Spikes are generally recognized by the GeneMapper ID X software and labeled as such automatically Dye artifacts may be present at numerous locations These artifacts typically do not have correct peak morphology though they may interfere with the interpretation of samples with a low amount of DNA iv Stutter peaks gt
55. e Instrument vi vii Remove the polymer 3500 POP 4 from the refrigerator allowing it to equilibrate to room temperature approximately 30 minutes Turn on the computer Turn on the AB 3500 Genetic Analyzer Login to the Windows Vista 3130User account Wait for the 3500 processes to load on the taskbar Open the AB 3500 Data Collection Software Using the dashboard monitor in the 3500 software determine which consumables need replenished and or replaced gt Replacement of buffer cartridges a Anode ABC and cathode buffer containers CBC may be replaced by simply removing the old ones and installing the new ones i Install a new CBC by peeling off the plastic film from the container and lock it into position on the instrument can only be installed the correct way due to the shape of the container The septa from the previously used container may be used if they appear to be in good shape If the integrity or age of the septa is in question replace both with new septa ii Install a new ABC by first tipping the container to maximize buffer volume in the main reservoir and 34 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice minimize the overspill reservoir v
56. e U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice This project was not a research project but addressed a critical need which was to establish a fully functioning CODIS dedicated laboratory by the procurement of dedicated CODIS Unit laboratory equipment Experimental design and approach The approach to this critical need based project is not research or experimental The problem was method development which calls for a more validation based design We chose a method development path based on establishing as automated a system as possible designed for a relatively small laboratory with medium to low throughput requirements Equipment was purchased following all federal and state purchasing guidelines AB Identifiler Plus kits were chosen to allow standardization of amplification kit between the Databasing and Casework laboratories This consistency is more efficient in sample ordering reagent QC and future training processes A direct amplification based method which does not require extraction or quantitation steps was chosen to reduce the amount of analyst steps necessary in the process as well as minimizing pre amplification reagent costs associated these steps The end results of the work product from this grant are the validation studies of the
57. e official position or policies of the U S Department of Justice were directly amplified from a 1 2mm punch wherein the template input DNA concentrations were not controlled or known Any samples demonstrating possible dropout homozygotes below 450 RFU or no result at locus or off scale data outside of amelogenin were removed from the sample analysis pool Average and minimum peak height ratios are calculated for each locus to determine the intra locus balance of each locus as well as a general robustness of the amplification kit as a whole Methods 109 convicted offender DNA profiles obtained in the concordance study were analyzed with thresholds established in the sensitivity stochastic background studies and stutter studies Manual data analysis was performed in the GeneMapper ID X software to omit any extraneous artifact peaks and the data sample marker alleles and allele heights was exported to an Excel worksheet for peak height ratio analysis All heterozygous loci were used in the comparisons whereupon the height of the smaller peak was compared to the height of the larger peak Ratio results were summarized as the percent height of the smaller peak to the larger peak Results The average peak height ratios ranged from 81 1 D2S1338 to 91 9 D5S818 The lowest peak height ratio observed for the Identifier Plus direct amplification method genotyped on the 3500 analyzer was 40 D18S51 The two lowest peak height ratio electrop
58. e the BSD humidification and dust collection systems v Activate the BSD software B Instrument and computer shutdown i Shut down the BSD software ii Turn off the BSD humidification and dust collection systems iii Turn the BSD power off iv Shut down the computer if desired C Plate and instrument preparation i If non FTA substrates are being sampled i e Bode buccal collectors the addition of Bode PunchPrep or AB Prep N Go is necessary to assist in the lysis process gt Vortex spin and pipette 2 0ul of Bode PunchPrep or AB Prep N Go into all wells of a new 96 well plate that will have non FTA substrate punches added gt Pre heat the heat plate to 70 C ii Due to the small size 1 2mm of the punches generated from the BSD robot static electricity must be minimized in the plate and instrument components most prone to harboring electrical charges e g plastic rubber components gt Use an ionization bar e g Amstat part 2U500 to neutralize charges on the 96 well plate and the lower components near 24 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice vi vii viii the plate tray of the BSD The most effective ionization occurs approximately 1 fro
59. ecked annually A reagent blank control is an analytical control sample that contains no template DNA and is used to monitor contamination from extraction to final fragment analysis This control is treated the same as and parallel to the forensic and or casework reference samples being analyzed A positive amplification control is an analytical control sample that is used to determine if the PCR performed properly This control consists of the amplification reagents and a known DNA sample A negative amplification control is used to detect DNA contamination of the amplification reagents This control consists of only the amplification reagents without the addition of template DNA Annually once per calendar year Materials and Reagents Materials i 9947 control DNA Refer to the Biology Unit Chemistry Manual and the Biology Unit Equipment Manual for specific information 5 Calculations N A 6 Uncertainty of Measurement N A 7 Acceptance Criteria A Refer to Databasing Technical Manual 6 for interpretation of reagent blanks positive quality control samples negative amplification control samples and positive amplification controls 8 Limitations N A 22 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position o
60. ed 10 additional alleles This unknown profile was keyboard searched in the offender database and matched to the sample 03F0884 which was one of the sensitivity samples This low level contamination might be attributed to the inexperience of the laboratory intern performing the dilutions and PCR setup as setup of the sensitivity study did not utilize QIAgility liquid handler automation An electropherogram of the 15 second injection has been included in the validation binder contamination assessment chapter AB 3500 ID Plus Validation Decreased Injection Time Summary Summary All sensitivity stochastic study samples were subjected to a decreased 4 second injection time N 32 In all cases excluding dropout events allele calls obtained from the decreased injection times were concordant with the 8 second standard injections A brief comparison of the 4 and 8 second electropherograms demonstrated an approximate average peak height decrease 87 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice of 50 when performing a 4 second injection after the original 8 second injection This study provides support for a decreased time injection when necessary e g off scale data AB 3500 I
61. epartment of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice vi Verify all sample types are correct in the Sample Type column Sample types may include Positive Controls e g 9947a 007 DNA Negative Controls e g RB s Amp Negatives Allelic Ladders and Samples vii Press the green Analyze button when all samples have been labeled with a sample type an analysis method panel and size standard Provide a unique project name e g group name and the software will save the project during and after analysis viii Samples successfully meeting WSCL_ evaluation criteria reference section 6 2 2 should be appropriately marked in the Specimen Category e g Convicted Offender This field will indicate whether or not the sample is for export to the database Interpretation Guidelines i Refer section 6 7 for additional information ii Off scale data should be interpreted with caution gt If off scale data is present in any locus with the exception of amelogenin the sample must be re injected re prepared or re amplified to confirm the DNA profile present in the off scale sample Note A shorter injection time may be used iii Artifacts gt Shouldering may occur in amelogenin and some loci Samples with shoulders that do not interfere with th
62. equirements stated in section 5 2 2 C Spectral calibrations must meet requirements stated in section 5 2 3 12 Limitations A Avoid exposing size standards and allelic ladders to light B Capillary array should be changed when the number of injections reaches 160 manufacturer injection warranty threshold or when the array is showing signs of failure e g broad peaks poor sizing C Avoid leaving an array filled with polymer exposed to air for more than 30 minutes 13 Safety A Caution Formamide is a teratogen and is harmful by inhalation skin contact and ingestion Use in a well ventilated area Use chemically resistant gloves and safety glasses when handling Refer to MSDS for handling B Safety precautions shall follow the WSCL Safety Manual 14 Report Writing A Refer to the WSCL DNA CE Technical Manual 77 and or 26 and the WSCL Biology Quality Assurance Manual 8 for DNA CE report writing guidelines 15 References and Exhibits Exhibit 8 Manufacturer s technical manual s data centrifuges 43 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Exhibit 9 Manufacturer s technical manual s data vortexes Exhibit 10 Manufacturer s technical manua
63. eterozygosity were intentionally chosen to obtain a larger net for the detection of dropout the four samples chosen are completely heterozygous at the core 13 loci though known data did not exist for amelogenin D2 and D19 Sensitivity Results Samples amplified with an original concentration of 0 2ng ul demonstrated an increased frequency of pull up peaks baseline artifact peaks elevated peak heights some off scale data observed in smaller loci and amelogenin and shouldering in some of the smaller loci see figure 14 Samples in the 0 1ng ul group had some pull up and artifact peaks no off Figure 15 First dropout observed in study 0 025ng ul sample set scale data observed though as a whole appeared to be much better quality data than the 0 2ng ul group All samples in the 0 05ng ul demonstrated quality electrooherograms in the absence of reproducible artifacts one spike observed in the OOFO069 sample The 0 025ng ul sample group demonstrated the first instance of dropout see figure 15 This 0 025ng ul sample group demonstrated good overall quality though the intra locus peak 74 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice height ratios were beginning to show
64. extraction and quantitation All convicted offender samples are currently being processed at WSCL using the CODIS laboratory established under this solicitation without additional staffing Average sample backlog has been reduced to less than 60 days from receipt until CODIS entry This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice IV Technical Report Introduction The Wyoming State Legislature funded the construction of a state laboratory building complex in Cheyenne Wyoming which was completed in November 2010 Included in the building complex is over one thousand square feet of space dedicated to CODIS laboratory and CODIS administrative functions Vestibule Casework Casework Extraction and Purification PCR Setup Vestibule Casework Amplification and Genetic Analyzer Typing 3130 Casework Screening Serology and DNA Preparation e i e Q l o eTo oO Post Amp Freezer Vestibule Casework D 5 Se Casework Examination Room 1 T Examination Room 2 cc ie Storage Figure 5 Layout diagram of the Biology DNA unit at the new Wyoming State Crime Laboratory This document is a research report submitted to th
65. f control or standard failure F CODIS Entry i Refer to CODIS Technical Manual Chapter 8 for acceptance criteria of convicted offender samples G Other issues not specifically addressed elsewhere may be evaluated as they arise In every case the reasoning of the involved parties must be documented and archived within the case file Sample interpretation for issues not specifically addressed in BDU manuals must be made with the consensus agreement of the analyst and the DNA Technical Leader Once an issue is identified consideration must be given to specifically addressing the issue in the Databasing Technical Manual or BDU QAM 8 Limitations A Peak height ratios that fall below 50 may be an indication of mixtures degraded samples etc B Low level and degraded samples may be susceptible to stochastic effects leading to allele dropout Care should be used when interpreting these samples 9 Safety A Safety precautions shall follow the WSCL Safety Manual 10 Report Writing N A 11 References and Exhibits A Exhibit 12 GeneMapper ID X software user manuals and data 12 Record Keeping A All documents will become part of the sample record and will be maintained in accordance with the LPPM 13 3 Record keeping for case records 53 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are t
66. f the validation new panels bins analysis methods quality flags and stutter thresholds were created specifically for this direct amplification procedure and have been found to be appropriate For more details see each chapter of the validation study AmpF STR Identifiler Plus System A The AmpF STR Identifiler Plus DNA typing system Applied Biosystems utilizes the polymerase chain reaction PCR to amplify regions of DNA known as short tandem repeats STRs in order to characterize DNA extracted from forensic specimens The AmpFfSTR multiplex systems allow for the simultaneous amplification of numerous STR loci as well as a portion of the Amelogenin gene located within the X and Y chromosomes Analysis of Amelogenin allows for gender determination The AmpFf STR Identifiler Plus kit contains the reagents needed for amplification including primer sets specific for the various loci the required allelic ladders and Amplitaq Gold DNA polymerase The locus specific sets consist of primers each labeled with one of four fluorescent dyes which are detected as different colors The use of multicolor dyes permits the analysis of loci with overlapping size ranges The amplified fragments are separated according to size by capillary electrophoresis CE and detected by laser excitation using an ABI PRISM genetic analyzer The reference allelic ladders for each of the STR loci and reference fragments for Amelogenin are also subjected to ele
67. fer Punches were sampled from the collectors as similarly as possible from the same region on the buccal collector The samples were treated identically and amplified with Identifiler Plus on the same sample plate Both sample sets were subjected to the same capillary electrophoresis conditions on an Applied Biosystems 3500 genetic analyzer and analyzed in GeneMapper ID X to determine if the new Prep n Go buffer is at least as effective as the currently used Bode PunchPrep treatment Results Electropherograms obtained from the previously described method were compared between each respective pair of samples and compared evaluated for dye specific balance locus specific peak height ratio and locus specific peak height amplitude 95 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Dye specific balance was evaluated by comparing the smallest fragment locus combined peak height of each specific dye channel to the respective dye s largest fragment locus combined peak height Though a theoretical perfectly balanced sample might demonstrate results around 100 the 9947a control sample which is not subject to confounding and possibly inhibiting factors that may be present on a buccal collection device
68. ffects Check the pump assembly for bubbles and run the remove bubble wizard if necessary 3500 User Guide page 251 Biology Exhibit 59 01 Add 297ul of Hi Di formamide and 3ul of G5 matrix standard to a microcentrifuge tube Briefly vortex and spin down contents Dispense 10ul of the master mix into each of wells A1 through H1 8 wells Cover the plate with a 3500 plate septa Denature the plate for 3 minutes at 95 C and then cool the plate on an ice block for 3 minutes 36 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice gt Inthe software click the Maintain Instrument button a Click on Spectral under the calibrate section in the left hand navigation pane b For number of wells select 96 c For plate position select A d Select Allow Borrowing e Select Matrix Standard from the chemistry standard menu f Select G5 from the drop down menu for dye set gt Load the plate into position A on the instrument and press Start Run gt Acceptance criteria for the spectral calibration are automatically evaluated in the software a If the calibration fails Reject Results and perform spectral calibration troubleshooting 3500 User Guide page 301 Biology Exhibit
69. flect the official position or policies of the U S Department of Justice vi vii viii Tri alleles Convicted offender samples Suspected tri alleles must be re amplified for confirmation before entry into CODIS Casework reference samples Suspected tri alleles should be interpreted with caution and may be re extracted and or re amplified Samples exhibiting dropout in one or more loci may be acceptable for database purposes provided that no dropout is suspected in any of the loci making up the current NDIS definition of core loci Samples with possible data below the threshold limit gt An additional injection of fifteen 15 seconds may be used for samples exhibiting possible data below the threshold limit A fifteen second injection time is not meant as a replacement for the standard eight second injection time An eight second injection time must always be performed and a fifteen second injection time may follow in those instances where its use is appropriate Results from fifteen second injection times must be interpreted with caution and the associated profile generated from the standard initial eight second injection time must be utilized in the analysis All associations and or identifications resulting from profiles generated with an increased injection time must be confirmed by the DNA technical leader i e casework reference samples Heterozygous single source profile alleles resulting from a
70. g the equipment and methodology described herein The current sample backlog as of 12 31 2011 is approximately 50 samples Or current sample volume is approximately 100 per month One quarter time analyst is easily able to process ninety samples per week using this methodology This has been critical to our laboratory s ability to keep the Offender backlog minimal without the loss of analysts from the casework laboratory We anticipate a large increase in Offender sample submissions when the State of Wyoming authorizes the collection of samples from all felon arrestees The capacity of this method will be able to be more accurately characterized when the sample volume increases We are also currently in the process of determining the viability of running casework reference samples through the efficient and automated CODIS laboratory system If the direct amplification system can be shown to produce acceptable and reproducible results we anticipate validating the methodology for casework reference sample applications Discussion of Findings 55 Vi This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice The goals and objectives of this project was to establish and increase the capacity of the WSCL COD
71. ginal injection The user may set this value at their preference Repeat these steps for additional assays 40 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice 5 Instrument Maintenance Wizards The 3500 instrument has multiple wizards to assist the user in performing step by step maintenance procedures The following wizards are available to the user A B T G Install Capillary Array for installing a new or used capillary array Remove Bubbles from Polymer Pump for removing bubbles in the polymer pump and or channels throughout the block Wash Pump and channels 40 minute procedure to wash polymer pump and channels with a new conditioning reagent Shutdown the Instrument procedure for long term shutdown of the instrument Fill Array with Polymer fills the array with fresh polymer Replenish Polymer primes the block and pump with new polymer displacing the previous polymer with the new Change Polymer Type used if changing from POP4 to POP6 POP7 6 Other Maintenance Procedures The 3500 user s guide Biology Exhibit 59 01 pages 230 232 details the maintenance procedures of the machine Consult this manual for detailed maintenance recommendat
72. hard stops At the point in the validation study prior to qualifying test after all foundational studies the 3500 capillary array has 114 injections and is still presenting good quality data with no or few broad peaks observed It should be noted that further use of the instrument beyond this validation study demonstrated that capillary arrays with over 400 injections can still produce good data with sharp well defined peaks 94 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Applied Biosystems Prep n Go Method modification validation Introduction The direct amplification of Bode buccal collectors has been facilitated by the addition of 2ul of Bode PunchPrep buffer which is purported to allow a direct amplification from a non FTA substrate Applied Biosystems has recently developed and marketed a buffer with the same goal of direct amplification of the untreated Bode buccal collectors The WSCL DNA unit will investigate this new buffer as a substitute for the Bode PunchPrep buffer Methods A set of six 6 previously run buccal collectors were punched using a 1 2mm Harris punch in duplicate with one set placed into 2ul Bode Punchprep and the other set placed into 2ul AB Prep n Go buf
73. he desired well Each sample will be followed by a cleaning punch which goes into a large trash can well beside the plate In chronological order from the beginning the import file for the BSD is loaded onto the computer applicable wells in the sample plate are pipetted 2ul of Bode PunchPrep the sample plate is irradiated the plate is installed on the front position of the BSD robot two 96 well positions exist on the robot samples are scanned and punched into their respective wells and the plate is removed from the BSD and installed on the heat block where it is incubated at 70 C for 20 minutes At this point the sample plate is ready for PCR setup B PCR Setup Though manual preparation and dispensing of master mix into the sample plate is allowed a protocol on the QIAgility liquid handler has been validated along with the study Through the preliminary stages of the validation the optimal PCR mix was found to be the manufacturer recommended 10ul of PCR Reaction Mix with 5ul of PCR Primer mix and 17 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice 10ul of water with the 1 2mm sample punch The 10ul of water was added in place of 10ul of DNA extract as the sample punch in the 10ul
74. he Identifiler Direct chemistry on a 3500 genetic analyzer Database Manager produces import files for the 3500 analyzer the BSD robotic sample puncher and can mark questionable sample locus results Validation Check Adding samples to queue Both automated and manual formats exist for adding samples into the queue database These samples add directly into the queue as ungrouped U samples Samples added through the automatic method have the prefix e g C11 for 2011 applied followed by a zero placeholders to create the five digit serial number These functions work as expected New group from queue The user is given an option to create a full plate of 90 samples or adjust the plate to a smaller size The counter is accessed which is housed in the queue database file and a new group is created with prefix G and a five digit serial 57 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice number one digit sequentially greater than the last group created The queue counter is updated and the sample group is created as per the user s request Any existing priority samples are applied to the new group followed by the oldest ungrouped queue samples This function works as expected Phas
75. he program handles the dynamic password internally and in real time so multiple instances of the program can successfully and securely access the databases These functions work as expected Miscellaneous Many other minor details of the program exist e g userboard for posting messages to other databasing users lot number database for recording applying current reagent lot numbers These functions have been tested and work as expected Conclusion 59 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice The Database Manager v2 0 has demonstrated reliable and reproducible worksheet generation queue sample group management and accurate date time user recordings of all phase related events Major as well as the minor program components not mentioned above work as expected Database Manager v2 0 is considered to be validated for use in routine forensic databasing applications 60 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Appendix B Wyoming
76. herograms D18S51 and D2S1338 were printed and saved in the validation binder peak height ratio chapter with applicable notes observations 84 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Conclusions The average peak height ratios obtained in this study demonstrate an overall good kit balance especially when the variables involved in the direct amplification process on buccal collectors are taken into account Peak height ratios below 50 were infrequent observed at only 2 of the 1333 loci used in this study and should be interpreted with caution in database and casework applications Notes Due to the concordance sample set used in this study being verified for true homozygosity at the D5 locus the population set for the peak height ratio study was 2 These two D5 loci available for comparison were detected as actual heterozygotes Though the population size was smaller than may be desired they both demonstrated good peak height ratios 96 9 and 86 8 which gives no support for concern about imbalance at the D5S818 locus Average Peak Height Ratios Across ID Plus Loci 8S1179 358 H01 317 539 D3S1 T D135 D16 1338 D19S433 vWA PO D21S11 xX D2 O co a R E X
77. hose of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Protocol 7 Analysis Parameters Analysis Notes 3500 with Identifiler Plus GeneMapper Analysis Settings GeneMapper Software Version ID X 1 2 AmpFLSTR Panels and Bins Version ax Marker Specific Stutter PHR s D8S1179 10 45 Heterozygous Peak Threshold 13 90 9 69 Homozygous Peak Threshold 450 RFU C caro 9 20 14 84 6 95 9 93 Smoothing and Baselining Method Light 9 93 11 53 Size Calling Method Local Southern Method 12 44 11 67 Polynomial Degree Minimum Peak Half Width i Peak Window Size Authorized By Bill Gartside Working Copy When Outside the LOAD Form 2833 Rev 1 0 Status Curent Paget oft Figure 12 Analysis parameters worksheet 54 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Results and Conclusions Statement of Results As this is not a research project there are no experimental results to report The end work products of this study are the protocols and validations studies presented elsewhere in this report As of 12 31 2011 the WSCL has processed approximately 1675 offender samples usin
78. ions A Daily with use procedures i Check for bubbles in the pump block and channels ii Check that the capillary tips are not crushed or damaged iii Ensure the pump block is in the pushed back position iv Clean instrument surfaces of dried residue spilled buffer or dirt v Check for leaks and residue around the buffer pin valve check valve and array locking lever Weekly procedures i Run the wash pump and channels wizard ii Use a lab wipe to clean the anode buffer container valve pin assembly on the polymer delivery pump 41 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice 7 Definitions A iii Restart the computer and instrument Monthly procedures i Flush the pump trap with purified water ii Check disk space iii Defragment the hard drive do not defragment the database drive iv Archive and remove old plates from the library DNA type or DNA profile is the genetic constitution of an individual at defined locations loci in the DNA A DNA type derived from nuclear DNA using STR technology typically consists of one or two alleles at several loci DNA technology is the term used to describe the type of forensic DNA analysis performed in the labora
79. ipment Manual for additional information i GeneMapper ID X v1 2 software 5 Calculations N A 6 Uncertainty of Measurement N A 7 Acceptance Criteria A Single Source DNA Samples i No off scale data present with the exception of amelogenin ii Only one or two alleles present at all loci examined with the exception of tri alleles iii The peak height ratios of heterozygote individuals at a locus should be 50 or greater Peak height ratios of less than 50 should be interpreted with caution 50 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice iv Stutter peaks should be within the expected values v Homozygous alleles must be above the stochastic threshold 450 RFU to be used for database eligibility statistical analysis Heterozygous alleles must both be above the analysis threshold 150 RFU to be used for database eligibility statistical analysis Mixed DNA Samples i Mixed DNA samples are not validated for use with the Identifiler Plus chemistry database system at this time Inconclusive allele calls n those cases where peaks are not clearly resolved and or the higher molecular weight alleles are not present due to degraded DNA allele calls
80. l s data pipettes Exhibit 11 Manufacturer s technical manual s data AB 9700 Thermal Cycler Exhibit 12 GeneMapper ID X software user manuals and data Exhibit 59 AB 3500 Genetic Analyzer technical manual s user manual s and data 16 Record Keeping A All documents will become part of the sample record and will be maintained in accordance with the LPPM 13 3 Record keeping for case records B Electronic files created by the AB 3500 Genetic Analyzer and from GeneMapper ID X software are permanently stored in the DNA Technical Leader archive file located on the DNA Technical Leader s M drive These files are write protected and are routinely backed up by the DCI IT department Access is limited to the DNA Technical Leader and the DCI IT administrator 17 Forms 3A Database Sample Worksheet 5A 3500 Maintenance Sheet 44 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Protocol 6 Interpretation with Identifiler Plus Interpretation with Identifiler Plus 1 Scope Raw data collected from the AB 3500 instrument must be analyzed and interpreted to be useful forensically This technical SOP governs the interpretation standards and guideli
81. le Note This data is based on the analysis of the same 18 allelic ladders used in the precision study AB 3500 ID Plus Validation Concordance Study Summary Introduction Using a direct amplification procedure with the Identifiler Plus PCR chemistry 109 convicted offender samples collected and archived on Bode buccal collectors were genotyped and the results were compared with their respective known DNA profiles from the CODIS database These comparisons were performed to determine the consistency and reproducibility of the procedures being validated Methods Over the course of three large amplification groups 21 36 and 52 samples respectively after filtering out samples with possible drop out and off scale data 109 offender samples were amplified and compared to their previously analyzed profiles 1 2mm punches were generated on the BSD 600 Duet in 96 well plates each with 2ul of Bode PunchPrep solution lytic assist for non FTA samples The PunchPrep sample plate was incubated for 20 minutes at 70 C as per the manufacturer s recommended procedure Sample plates were placed on the QIAgility liquid handler for PCR setup Added to each sample well was 10ul water 10ul ID Plus reaction mix and 5ul ID Plus primer mix Plates were sealed and amplified on the 9700 thermal cycler as per ID Plus 71 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department
82. lect the official position or policies of the U S Department of Justice AB 3500 ID Plus Validation Qualifying Test Summary Methods Eleven different WSCL convicted offender buccal collector samples with known profiles were obtained Each collector was sampled with the BSD 600 Duet sample puncher as a 1 2mm punch The QIAgility liquid handler was used to perform the PCR setup for the plate with the Identifiler Plus Chemistry and the sample plate was amplified on a 9700 thermal cycler Following amplification the samples were genotyped on the 3500 genetic analyzer Data analysis was performed with GeneMapper ID X v1 2 Results All samples genotypes returned profiles correctly matching their respective known profiles The D2S1338 and D19S433 loci were not previously profiled and were therefore not available for comparison with known data Negative and positive controls produced the expected results Two samples demonstrated possible dropout on the first 8 second injection application A second run was created with the appropriate controls and an increased 15 second injection was applied to two samples and their associated reagent blank Though one sample C0701360 would have failed on stochastic grounds actual homozygous peak at CSF with 8 second injection was only at 328 RFU all samples demonstrated perfect concordance with their respective known profiles Conclusion These results support the conclusion that the protocols in use at
83. lled and the applicable protocols are initiated Standard injection time on the 3500 is 8 seconds though the validation supports the use of increased and decreased time injections Import files that contain sample well positions sample names and desired protocols are able to be created and imported to the 3500 software which will likely be used more frequently than manual data entry on the 3500 Analysis GeneMapper ID X software has been validated for use with data analysis at the WSCL In the course of this validation new panels bins analysis methods quality flags and stutter thresholds have been created specifically for this direct amplification procedure and have been found to be appropriate For more details see each chapter of the validation study 68 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice AB 3500 ID Plus Validation Precision Study Summary Introduction Allelic ladders for the Identifiler Plus kit were injected on the AB 3500 and analyzed with GeneMapper ID X software to determine the precision of size calls for the instrument and procedure used by the WSCL Methods Two different allelic ladder plates were prepared according to the manufacturer s protocols for the form
84. lowing studies will be explicitly performed in this validation Precision reproducibility concordance sensitivity stochastic background stutter peak height 61 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice ratios increased injection time and decreased injection time Further assessments of contamination and non probative evidence will be made along with a qualifying test for the system chemistry Methods Results Discussion Conclusions See each study s individual section for the section summary and associated data The majority of raw data electropherograms will be maintained in an electronic archiving method to preserve paper and the size of the validation binder Outline of criteria from Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM 1 General Considerations for Validation of the DNA Analysis Procedure 1 2 2 2 Internal validation should lead to the establishment of documents quality assurance parameters and interpretation guidelines Precision Study Adopted 0 5 base pair bin from Applied Biosystems Sensitivity Study Stochastic Limit 450 RFU and DNA target amplification amount of 0 05 ng uL to 0 1 ng L Background S
85. lyse the cells prior to PCR setup A product developed from Bode Technologies Bode PunchPrep requires 2ul of PunchPrep per well with a 1 2mm punch to be incubated at 70 C prior to PCR setup thereby transforming the filter paper into something functionally similar to an FTA substrate Due to the small size of the punches and the potential effects of static electricity from a plastic 96 well plate the plastic sample plates are subjected to brief irradiation with a 500 microcurie alpha particle emitter Amstat Industries part 2U500 which ionizes the plastic with both positive and negative charges The plastic 96 well plate with static electricity has a build up of negative charges whereupon ionization will allow the plate to take up positive charges neutralize 65 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice the static potential and allow small sample punches to rest in the bottom of the wells without jumping or sticking The BSD 600 Duet sample puncher is loaded with a file containing all expected barcodes on the plate generated prior to sample punching Sample barcodes are scanned and the BSD moves to the correct sample well incorrect and or out of place barcodes will result in an error
86. m the bar gt Slowly apply ionization to the front and back of the 96 well plate followed by the front plate tray inside the BSD as well as the spot detector s rubber cover BSD plate setup Apply an input file to the desktop for the desired sample group for punching Delete any previously used plate files on the computer s desktop Execute the BSD Duet Menu program from the shortcut on the computer s desktop Press the Distribute Spots button to execute the collection software At this point the BSD robot should move around to test its axes and check the performance of the spot detector If the spot detector passed its checks press continue to move on gt If the software insists that the spot detector is not working properly close the software turn off the BSD turn on the BSD and enter the software again If this problem is not resolved by a restart the spot detector connections may need to be physically verified in the lower part of the BSD instrument Press continue on all available tests ensure selection of the Front test checkbox and have the checkboxes Samples and Cleaning checked Press Continue Load the 96 well plate previously ionized to prevent static charges from interfering with punch collection into the front plate tray in the BSD Press Continue Check the cleaning well to ensure that it is not filled and empty it if necessary Press Continue
87. mple set demonstrated on average higher quality electropherograms see figures 25 26 In the course of databasing at the WSCL large fragment loci on an otherwise good profile have routinely demonstrated low level and or stochastic effects With the addition of the AB Prep n Go buffer some of these effects may be avoided ultimately increasing the first and second pass rates of database samples through the system indirectly saving costs on rerun associated consumables and analyst time These results also support an expectation of average increased electropherogram quality 98 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Figure 25 Sample result using Bode PunchPrep buffer Figure 26 Sample result using Applied Biosystems Prep n Go buffer same sample as in figure 25 99 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Average Peak Height Change with Prep n Go Buffer vs PunchPrep 600 00
88. n Identifiler Plus PCR Amplification kit 8 Calculations As described in 4 3 2 above 9 Uncertainty of Measurement N A 10 Acceptance Criteria A Refer to the Databasing Technical Manual 6 for interpretation of reagent blanks positive quality control samples negative amplification control samples and positive amplification controls 11 Limitations A The fluorescent dyes attached to the primers are light sensitive Store the primer sets and amplicons protected from light B Amplicons may be stored at 2 to 8 C for up to 7 days or at 35 to 0 C for extended periods 32 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice C Long term storage of amplified samples at 4 C or higher may produce degradation products 12 Safety A Safety precautions shall follow the WSCL Safety Manual 13 Report Writing N A 14 References and Exhibits A Exhibit 7 Manufacturer s technical manual s data hoods Exhibit 8 Manufacturer s technical manual s data centrifuges B C Exhibit 9 Manufacturer s technical manual s data vortexes D Exhibit 10 Manufacturer s technical manual s data pipettes E Exhibit 11 Manufacturer s technical ma
89. n increased injection time may be used for database entry inclusion or statistical purposes if applicable Homozygous alleles that are moved above the stochastic threshold with an increased injection time may not be used for 49 3 Definitions A This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice database entry or for statistical purposes All homozygous alleles characterized with an increased injection time may be used for exclusion purposes A reagent blank control is an analytical control sample that contains no template DNA and is used to monitor contamination from extraction to final fragment analysis This control is treated the same as and parallel to the forensic and or casework reference samples being analyzed A positive amplification control is an analytical control sample that is used to determine if the PCR performed properly This control consists of the amplification reagents and a known DNA sample A negative amplification control is used to detect DNA contamination of the amplification reagents This control consists of only the amplification reagents without the addition of template DNA 4 Equipment Materials and Reagents A Equipment Note Refer to Biology Equ
90. n 60 days from receipt until CODIS entry This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Table of Contents Abstract Table of Contents Executive Summary Technical Report a b C d e f g Protocol 1 Method Introduction Protocol 2 Method QC Measures Protocol 3 Sample preparation with the BSD Puncher Protocol 4 Direct Identifiler Plus Amplification Protocol 5 AB 3500 Genotyping Protocol 6 Interpretation with Identifiler Plus Protocol 7 Analysis Parameters Results and Conclusions Appendix A Software Validation Appendix B Method Validation Summaries Precision Study Reproducibility Study Concordance Study Sensitivity Study Background Study Stutter Study Peak Height Ratio Study Non Probative Evidence Study Contamination Assessment Decreased Injection Time Increased Injection Times Validation Notes AB Prep and Go Buffer Addendum Page 1 Page 3 Page 4 Page 9 Page 16 Page 21 Page 24 Page 29 Page 34 Page 45 Page 54 Page 55 Page 57 Page 61 Page 69 Page 70 Page 71 Page 73 Page 78 Page 81 Page 83 Page 86 Page 86 Page 87 Page 88 Page 90 Page 95 This document is a research report submitted to the
91. n in Wyoming Both of these sample collector types have been considered to be candidates for the direct amplification system currently in validation and may be integrated into the validated procedure at a later date pending the acceptance of a future validation check AB 3500 ID Plus Validation Contamination Assessment Summary Introduction All DNA samples amp positive controls amp negative controls and reagent blanks from each of the previously conducted validation studies were evaluated for the correct genotype or absence thereof 86 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Results None of the samples or controls in the validation study demonstrated detectible contamination at the 150 RFU detection limit employed from the results of the background study Conclusions These results support the conclusion that the procedure used in this validation process provides sufficient protection from cross contamination Notes An amplification negative sample in the sensitivity study injected with an increased injection time 15 seconds was determined to have a low level of contamination present due to the presence of an amelogenin X The analysis method was modified to 60 RFU which obtain
92. nes used by the WSCL biology unit 2 Procedure A GeneMapper ID X Software Setup GeneMapper ID X is a networked system that utilizes a central database to manage panels projects analysis methods size standards plot settings and more Due to the controlled nature of the software available analysis methods panels and size standards are identical on all computers connected to the same host database copy computer Select the Add samples to project button alternatively use the file menus Edit gt Add samples to project ii Navigate to and add all appropriate relevant samples to the new project ili Under the Analysis Method column select ID Plus_Databasing_ 3500 and apply to all applicable samples in the project gt As per the validation study the following thresholds have been established a Detection threshold at 150 RFU b Stochastic threshold at 450 RFU gt The analysis range may need adjusted depending on the position of the actual data The analysis method may be edited to expand shrink or shift the analysis range as long as the necessary sizing peaks 80 400 are in the analysis range v Under the Panel column select Identifiler_ Plus Panels_v1X and apply to all applicable samples in the project v Under the Size Standard column select CE_G5 HID GS600 and apply to all applicable samples in the project 45 This document is a research report submitted to the U S D
93. ng test may be administered internally externally or collaboratively AB 3500 ID Plus Validation Databasing System Procedure Introduction The State of Wyoming collects a DNA sample from all persons convicted of a felony level crime reference W S S 7 19 403 a The WSCL 64 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice complies with this law by distributing Bode buccal collector kits to law enforcement agencies and the Department of Corrections to collect reliable DNA samples and background information from the subject Collected samples are logged tested for a DNA profile reviewed and uploaded to an appropriate database as part of a national and state participation in CODIS Substrate Sampling Pre PCR Treatment Bode buccal collectors are sampled via a 1 2mm punch on a semi automated BSD 600 Duet sample puncher Direct amplification methods typically require samples from an FTA paper which are theoretically lysed cells with the DNA bound to the paper As the buccal collectors used at the WSCL are only a filter paper Bode purchases the filter paper from Whatman as per correspondence with GE Whatman representative Betsy Moran February 2011 an additional reagent is required to
94. nt analyst is signed 58 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice into the program Group notes are still accessible to the analyst for modification if necessary This function works as expected Technical Review When groups are actually technically reviewed the reviewer can select the appropriate group and push technical review from the review tab This will stamp the group sample worksheet with the date move the entire group folder and contents to the Archive Records folder and take all the group samples from the queue to the completed database Error checking functions insure that no files are open prior to moving the group folder These functions work as expected Program Control Security Initialization of the program results in a log on screen Users which can be modified by another user with administrative privileges must present their username and password to the logon screen The credentials are checked against a database containing all usernames and passwords and the user is either allowed or denied program access User passwords are protected in a password protected database that changes its password to a 10 digit random code each time a user successfully logs into the system T
95. nual s data AB 9700 Thermal Cycler F Exhibit 61 01 Manufacturers technical manual s data Qiagen QIAgility liquid handler G Exhibit 27 02 Applied Biosystems AmpFfSTR Identifiler Plus PCR Amplification Kit User Guide Part number 4402743 15 Record Keeping A Though QIAgility PCR setup records for databasing groups are not required to be included electronically or in hard copy with the databasing group records the record s should be saved locally to the QIAgility instrument in the event the record s are required for review troubleshooting or auditing purposes Applicable equipment and lot numbers shall be recorded on the database sample worksheet for record keeping with the respective databasing group 33 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Protocol 5 AB 3500 Genotyping Genotyping on the AB 3500 Genetic Analyzer 1 Scope The ABI PRISM 3500 Genetic Analyzer is a capillary electrophoresis platform used to generate DNA profiles This technical SOP governs the use of the AB 3500 instrument and its associated collection software for generating DNA profiles for genetic typing using STR technology 2 Instrument Setup Procedure A Setting up th
96. of Justice vi vii viii Xi xii xiii Xiv XV Combine size standard and Hi Di formamide in a 1 5ml microcentrifuge tube using the following formulation gt Number of samples x 0 5ul of LIZ size standard gt Number of samples x 8 5ul formamide Dispense 9ul of the formamide LIZ master mix into the appropriate wells on a 96 well plate Vortex and spin down the appropriate allelic ladder tube Dispense 1ul of allelic ladder or PCR product amplicon according to the recorded plate layout Place a new 3500 plate septa on the plate Place the plate in the 95 C thermal cycler for 3 minutes Place the plate on ice for at least 3 minutes Install the plate into a plate base and cover with a plate retainer Ensure the plate retainer and septa strip holes align correctly Press the tray button on the 3500 and load the prepared plate Close the instrument doors and press the button Link Plate for Run Verify the plate s in positions A and B Press Start Run 4 Sample Reinjections A Samples may need to be reinjected due to partial profiles off scale results allelic confirmations or sizing quality To perform a reinjection Select the samples requiring a common assay Click the Re Inject button on the top of the screen Select the Reuse a protocol in the library option and choose the desired assay Placement of re injections may be following all injections or after ori
97. of dedicated CODIS Unit laboratory equipment Wyoming Offender samples are currently being collected and archived with Bode buccal collectors The method that was validated and is currently in use at the WSCL for convicted offender sample processing uses BSD punchers for the robotic placement of 1 2mm punches in a 96 well plate Punches are directly amplified without extraction or quantitation with Applied Biosystems AB Identifiler Plus kits Amplification set up is performed using a Qiagen QIAgility robot and amplification is performed in an AB 9700 thermal cycler Analysis is performed using an AB 3500 eight capillary genetic analyzer and AB GeneMapper ID X data analysis software The process is managed by Database Manager an internally developed Excel based Visual Basic for Applications VBA information management system which facilitates all aspects of the methodology The current first pass success rate for convicted offender samples is greater than 95 Samples which are not initially successful and difficult samples are re routed through the WSCL Casework DNA laboratory for a more conventional analysis which includes DNA extraction and quantitation All convicted offender samples are currently being processed at the WSCL using the CODIS laboratory established under this solicitation Sample backlog has been significantly reduced The turnaround time from sample receipt to database entry has improved from more than 2 years to less tha
98. ogeneity have been reported in the literature and have been observed through practical experience in the laboratory These peaks will have a similar intensity to the other major peak for that locus but will not line up with the allelic ladder Alleles one two or three nucleotides shorter than the common four base repeat alleles cause the amplified allele to migrate faster than that standard allele in the allelic ladder An example of this is the common THO1 9 3 allele A rare microvariant will be described as the lower molecular weight allele designation followed by an x with x representing the number of bases greater than the lower molecular weight allele Rare variants will not be associated with a bin or virtual bin within the analysis software An allele located outside the range of the allelic ladder will be documented as lt or gt the largest or smallest allele for that locus Example an allele which migrates above the largest allele for the D16 locus will be documented as gt 15 Convicted offender samples Rare variants must be confirmed by re injection of the sample Casework reference samples Rare variants should be interpreted with caution and should be re injected 48 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily re
99. olume Carefully remove the film from the container to avoid spilling and install on the instrument with the RFID tag facing backwards to instrument interior gt Replenish Polymer gt a When necessary open the instrument door and remove the conditioning reagent or used polymer by moving the polymer lever down b Remove the film from the new polymer pouch being careful to not leave pieces of the film plastic in the pouch opening c Install the new polymer in the polymer head fitting The RFID tag for the polymer should be facing backwards Lift and secure the polymer lever to its original position Installation of capillary array a Close the instrument door Press the Tray button b From the Maintenance Wizards screen click Install Capillary Array c Follow the prompts in the given in the Capillary Array Wizard d Perform a spatial calibration Section 5 2 2 Spatial Calibration A spatial calibration should be performed whenever the capillary array has been moved the detection cell has been opened or the machine has been moved gt From the maintenance menu select Spatial Calibration from the navigation pane Select Fill or No Fill fill the array with polymer or not and click Start Calibration The calibration should show 8 approximately even sharp peaks with one marker at the top of every peak If the results meet these criteria select Accept Results Otherwise Reject Res
100. ontrol sample 51 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice vi Identifiler Plus Loci 9947 D8S1179 D21S11 D7S820 CSF1PO Amplification Negative Control No typed alleles present If the negative amplification control exhibits a DNA profile at a specific locus or loci any sample s concurrently typed with this control are considered inconclusive When run anomalies spikes dye blobs etc or other non amplification issues affect the interpretation of positive amplification control samples the control samples may be re analyzed separately from their associated samples If they meet the interpretation guidelines of section 13 7 5 upon re analysis their original associated samples are considered valid 52 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice vii If a control or standard does not meet interpretation requirements it must be re injected re prepared or re amplified based on analyst discretion and the nature o
101. r policies of the U S Department of Justice 9 Safety 10 11 12 13 A Safety precautions shall follow the WSCL Safety Manual Report Writing N A Record Keeping A All documents will become part of the case record and will be maintained in accordance with the LPPM 13 3 Record keeping for case records References and Exhibits A Exhibit 4 The FBI Quality Assurance Standards for Forensic DNA Testing Laboratories most recent version B Exhibit 5 The FBI Quality Assurance Standards for DNA Databasing Laboratories most recent version Forms N A 23 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Protocol 3 Sample preparation with the BSD puncher Sample Preparation with the BSD 600 Duet 1 Scope Protocol for the semi automated sampling of biological substrates into a 96 well plate for databasing applications 2 Methods and Controls A Instrument and computer activation i Start the computer ii Turn the BSD power on iii Check and adjust water in the humidification sponges if necessary gt Sponges in humidification system should be completely saturated with purified water with little or no standing water in the bottles iv Activat
102. re those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice AB 3500 ID Plus Validation Background Study Summary Introduction Eight negative controls were amplified with the Identifiler Plus chemistry each injected four times and analyzed to determine the detection threshold for the current procedure on the AB 3500 genetic analyzer Methods Eight negative controls were amplified for 28 cycles under the current validation procedure These control samples were set up with LIZ 600 formamide and analyzed on the 3500 genetic analyzer standard 8 second injections Each sample was injected in quadruplicate and two additional negative controls from the plate were included in the analysis A new analysis method was setup in GeneMapper ID X to analyze the blue green yellow and red dyes at 1RFU One of the sample injections amp control negative 6 hada bad injection and was removed thereby providing a total of 33 negative control electropherograms for the background analysis study All peaks in each dye set B G Y R gt 1 RFU were counted and averaged prior to calculating dye specific standard deviations and determining the highest background peaks observed in each dye channel A few non reproducible spike artifacts were removed after data analysis sample and artifact data are available on printed sheets in the background chapter of the validation binder Results The average peak
103. reflect the official position or policies of the U S Department of Justice sample processing method are termed phases These phases can be described as follows o Phase 0 Group members defined no physical work performed on any samples Sample database updated with phase 0 date time stamp o Phase 1 Group worksheet see figure 10 created with current reagent lot numbers and equipment BSD and 3500 export files created and submitted to networked group folder date applied to worksheet for BSD punching into the group plate sample incubation PCR setup and amplification Sample database updated with phase 1 date time stamp o Phase 2 Dates applied to worksheet reflecting 3500 genetic analyzer initiation with the respective group Sample database updated with phase 2 date time stamp Analysts will complete genetic analyzer run applicable reinjections and preliminary data review during this phase o Phase 3 Summary sheet creation and data check from GeneMapper ID X export file Summary sheets are saved in the respective group folder with color coded indicators flagging any questionable results e g more than two alleles at a locus possible contamination or sample duplication lt 50 peak height ratios Sample database updated with phase 3 date time stamp o Phase 4 Locked for technical review Group is transferred to review status which allows users other than the analyst submitting the group for review to technically review the g
104. research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice e Logs current reagent lot numbers equipment numbers and logs analysis dates on group record worksheets e Generates a group import file which automates sample plate creation on the AB 3500 genetic analyzer e Locks samples for technical review following analyses e Creates and checks all sample allele table results for the presence of contamination sample duplication peak height ratio discrepancies and allele counts greater than two e Manages reanalysis of samples that show unsatisfactory results or require additional analysis such as tri alleles Sample Preparation e The sample queue is updated with the most recent sample information When a group is initiated from this queue in a 96 well plate format a visual group worksheet is created and an export file compatible with the BSD 600 is generated and imported on the BSD in preparation for sample punching Figure 2 BSD Duet 600 e All convicted offender samples are scanned by the BSD 600 sample puncher figure 2 and punched into their respective wells in the plate Reagent and amplification controls are in pre programmed plate positions left empty at this point in the process Sample Extraction
105. roup samples see figure 9 Sample database updated with phase 4 date time stamp 12 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Database Manager 2 0 General Database Info Current Lot Numbers Databasing Functions Reviewer Functions Database Queue Group Sample Maintenance System Cor m Reagents QAMS Equipment Prep Buffer 010413 BSD 600 Duet Identifiler Plus 091612 Heat Block Water 011113 QIAgility Formamide 110312 Thermal Cycler LIZ 600 v2 022813 3500 Analyzer POP 4 Polymer 041212 Anode Buffer 040812 Cathode Buffer 040112 Logged in user smcwil Exit Datase Manager v20 Figure 7 Reagent lot and equipment management interface Database Manager 2 0 General Database Info Current Lot Numbers Databasing Functions Reviewer Functions Database Queue Group Sample Maintenance System Cor _ lt Group amp 00057 Status Phase 0 m Databasing Functions Group Notes Start DNA Phase 1 Start DNA P J Start DNA Phase 3 Submit For Initiate BSD punching PCR setup and amplification Options Reroute Failures Active Group Folder Logged in user smewil Exit Database Manage v20
106. s of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Methodology validation and protocols Appendix B is the validation study for the methodology direct amplification of a 1 2 mm punch from a Bode buccal collector using the AB Identifiler Plus amplification kit with electrophoresis on an AB 3500 eight capillary genetic analyzer The work products of this validation are the validated protocols used by the WSCL for offender sample processing The WSCL databasing technical manual Standard Operating Protocols are presented below Protocol 1 Method Introduction 1 Introduction to the Database System ID Plus 3500 Background The database system outlined here was designed as a solution to higher throughput DNA databasing testing without requiring a significant increase in staffing This system was purchased as the result of a generous grant from the NIJ in 2010 which was implemented and validated in the first half of 2011 Technology The database system takes advantage of a direct amplification system which effectively removes the labor time intensive steps of extraction and quantitation Sample punches are mixed with a PCR mix amplified directly in the reaction well and genotyped Casework Integration The database system has been designed to profile the majority of DNA samples in an efficient and timely manner Samples presenting difficulty
107. s when an allele comparing program was initiated Upon manual investigation the cause of the finding was due to the CODIS acceptable value of lt 12 not exactly matching the value of 11 in the validation data though they are equivalent 72 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice All positive controls i e 9947a from all studies performed were concordant at all tested alleles with the published control genotype AB 3500 ID Plus Validation Sensitivity Study Summary Introduction Four different convicted offender samples with known DNA profiles were serially diluted amplified and analyzed according to the current procedure in validation Samples were analyzed to determine the stochastic threshold and sensitivity levels Though the system is a direct punch amplification and does not use a quantitative template concentration this study will serve as evidence of the Identifiler Plus amplification kit sensitivity levels and Figure 14 Shouldering example at the D3S1358 locus 0 2ng ul assist in establishing a stochastic threshold for analysis of the direct amp samples Similarly this study will also provide an optimal template concentration range in the even
108. t false homozygote being detected at 427 RFU and the next highest detected at 385 RFU a 450 RFU stochastic threshold will be adopted for this procedure s data analysis protocol Samples with homozygotes below the 450 RFU range tend to exhibit greater peak imbalance Notes Based on these studies the most sensitive loci in the ID Plus kit are in order D19 vWA D13 D3 and D8 Loci most susceptible to dropout are in 76 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice order D7 CSF D18 FGA and D21 Samples used in these studies were also subjected to alternate injection times Total Dropout Observed by Locus mo v gt ie o wn 2 O gt o a o ie a 4 o v iT S vo o pe v a Figure 17 Sensitivity study results by locus dropout per locus Dropout Observed by Template DNA Concentration Percentage Dropout Observed 1ng 0 5ng 0 25ng 0 125ng 0 063ng 0 031ng 0 016ng Figure 18 Sensitivity study results by concentration dropout by concentration count in alleles 77 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed a
109. t samples are manually extracted and integrated into this procedure at the PCR setup step Methods The four known convicted offender profiles 00F0069 02F0539 02F0771 and 03F0884 used in this study were extracted purified by the EZ1 non differential method and quantitated with the Quantifiler Duo RT PCR kit per the current WSCL protocols all samples demonstrated quantitative values between 4 ng ul and 9 ng ul Each sample was diluted to 0 2ng ul and further serially diluted to concentrations of 0 1ng ul 0 05ng ul 0 025ng ul 0 0125ng ul 0 00625ng ul 0 003125ng ul and 0 001563ng ul 10ul of template DNA was 73 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice used in each amplification reaction full volume ID Plus reaction as per manufacturer recommendation All amplifications were injected on the AB 3500 instrument GeneMapper ID X software was used to analyze the samples with an analysis threshold of 150 RFU as was determined from the background study conducted prior to this study Though the original plate setup on the 3500 contained 4 8 and 15 second injections of the sensitivity study samples only the standard 8 second injections will be analyzed in this study Samples with high h
110. th a 1 2mm punch to be incubated at 70 C prior to PCR setup thereby transforming the filter paper into something functionally similar to an FTA substrate Due to the small size of the punches and the potential effects of static electricity from a plastic 96 well plate the plastic sample plates are subjected to brief irradiation with a 500 microcurie alpha particle emitter Amstat Industries part 2U500 which ionizes the plastic with both positive and negative charges The plastic 96 well plate with static electricity has a build up of negative charges whereupon ionization will allow the plate to take up positive charges neutralize the static potential and allow small sample punches to rest in the bottom of the wells without jumping or sticking The BSD 600 Duet sample puncher is loaded with a file containing all expected barcodes on the plate generated prior to sample punching Sample barcodes are scanned and the BSD moves to the correct sample well incorrect and or out of place barcodes will result in an error message on the computer regarding sample number expectations In the case of a correct sample scan the BSD will position the plate below the sample punch chute and activate the punch head The database analyst will position the buccal collector sample under the punch spot a red laser dot gives precise position of the area to be punched and will activate the BSD to execute the punch The punch will fall through the chute and into t
111. the WSCL are accurate and reproducible for genotyping DNA samples 89 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice AB 3500 ID Plus Validation Validation Notes BSD Optimization The BSD 600 Duet was purchased and installed through Applied Biosystems a new vendor of the BSD robotics The BSD sample puncher was initially tested with unstained filter paper for accuracy in placing samples into their desired wells These tests were somewhat frustrating as the static charges on the 96 well plates generally inhibited paper punches from entering the wells A few major corrections were implemented to optimize this system 1 Plate adjustment The configuration module of the BSD software was used to adjust the punch chute to be centered above the desired well Both BSD robots purchased were significantly off center and adjusted Full test plates were punched as a probationary test of the machine s accuracy and the success rate was greatly enhanced 2 Humidification system adjustment The humidification system installed with the BSD robots included an air pump with an adjustment dial on the back The included air pump is basically similar to an aquarium air pump for water oxygenation though it forces
112. the desired import file Click the Assign Plate Contents button to view the plate contents Verify the plate setup assays file naming and results groups are correct Create a manual plate record gt gt gt gt Click Create New Plate from the main dashboard view Input all information necessary into the designated fields a Name of plate must be a plate name unique to the library b Plate format select 96 well c Plate type select HID d Capillary length select 36 cm e Polymer select POP4 f Owner name of person running 3500 g Barcode optional field h Description optional field Click the Assign Plate Contents button on the bottom of the screen Type in all sample names in their respective plate locations 38 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice gt Click the Add Assay from Library link in the add assay box to add appropriate assays to the plate a Available assays include i IFP Norm_POP4_8s ii IFP Norm_POP4_15s iii IFP Norm_POP4_4s b The standard injection time for direct amplification samples with Identifiler Plus chemistry 28 cycles is 8 seconds 15 second injec
113. the single pipetting methods though it is a bit of a challenge on the QIAgility When a user without multi dispense pipetting experience on the QIAgility sets up a run on the QIAgility with multi dispensing they will discover a resultant plate with a shocking amount of variability The QIAgility has multi dispensing options such as include air in ejection a pre dispensing ejection volume an extra amount of volume to carry per sample during dispensing an extra volume per ejection amount and an ejection speed value Food coloring dye and water was used to make artificial reagents in the exact protocols used to perform the direct amplification PCR setups Through the testing and optimization of the preferences in the multi dispensing menu approximately 20 30 dye plates were prepared and evaluated for modification and or acceptance of the preference values Ultimately a protocol was generated with the multi dispensing options to create a uniform plate with a 25ul reaction in each well Error Minimization Steps A few different steps were integrated into the process in attempt to standardize the process and minimize errors 1 Cleaning punches are a different color than the generally white sample punches This is a quick visual aid to determining if there has been a sample misplacement or an inadvertent cleaning punch 92 This document is a research report submitted to the U S Department of Justice This report has not been published
114. tions and 4 second injections are available in the event that off scale or low level data is obtained To add a file naming convention click the Add from Library link in the file naming convention box a Add an appropriate _ file naming convention e g CODIS_FileNaming to the plate To add a results group click the Add from Library link in the results group box a Add an appropriate results group e g CODIS_ResultsGroup to the plate Select all appropriate samples in the plate and apply an assay file naming convention and results group Ensure all applicable samples have all three of these attributes Expand the Customize Sample Info box on the lower right of the screen Select allelic ladders and controls labeling them as such in the sample type drop down menu All plate wells are labeled as sample by default Save the plate by clicking Save on the menu Prepare the physical plate i Initialize pre heat a 95 C denaturing protocol on a thermal cycler ii Ensure the formamide aliquot is thawed and ready to use iii Vortex the GeneScan 600 LIZ v2 size standard and spin down ina microcentrifuge 39 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department
115. to the sample plate in preparation for sample genotyping This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice e After denaturing and snap cooling steps sample plates are added to the Applied Biosystems 3500 instrument figure 4 The 3500 injects eight samples at a time requiring approximately sixteen hours for two 96 well plates 24 injections as the eight capillary 3500 analyzer can hold two plates at a time e Raw data from the AB genotyping equipment is transferred to workstations where analysts manage the system software GeneMapper ID X to analyze and technically review data The review functions available in the ID X Figure 4 Appiled software assist in the technical review of data The Biosystems 3500 analyzed and reviewed data is finally exported from GeneMapper ID X to a Common Message Format cmf file and uploaded to the CODIS database Rerun samples are marked as such and resubmitted to the sample queue in Database Manager The current first pass success rate for Offender samples is greater than 95 Samples which are not initially successful and difficult samples are re routed through the WSCL Casework DNA laboratory for a more conventional analysis which includes DNA
116. tory such as RFLP STR Y STR or mitochondrial DNA Platform is the type of analytical system used to generate DNA profiles such as capillary electrophoresis real time gel and end point gel instruments or systems 8 Equipment Materials and Reagents A Equipment Note Refer to Biology Equipment Manual for additional information i AB 3500 Genetic Analyzer ii 9700 GeneAmp PCR System iii GeneMapper ID X v1 2 software iv Pipettes v Microcentrifuge vi Vortex Materials Note Refer to Biology Chemistry Manual for additional information i 3500 POP 4 ii Nanopure purified water iii Anode and Cathode Buffer Containers iv LIZ Size Standard v2 GS 600 v Identifiler Plus allelic ladder 42 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice vi Microcentrifuge tubes vii 96 well plate viii Deionized formamide 9 Calculations As described in 5 3 5 above 10 Uncertainty of Measurement N A 11 Acceptance Criteria A Refer to DNA CE Manual 70 for interpretation of reagent blanks positive quality control samples negative amplification control samples positive amplification controls and forensic sample data B Spatial calibrations must meet r
117. ts All sample wells contain a 1 2mm punch and Identifiler Plus master mix Through preliminary validation plates the optimal number of cycles was found to be 28 which is the cycle number on the Identifiler Plus Database protocol on all applicable thermal cyclers The sample plate is installed on the thermal cycler and the protocol is initiated which takes approximately 3 hours 67 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Genotyping on 3500 Analyzer Frozen formamide is thawed in preparation for genotyping on the 3500 The LIZ v2 0 size standard is removed from the refrigerator and mixed in proportion with the formamide to create the formamide LIZ master mix This master mix is applied to all applicable wells in a new 96 well plate in the correct volumes manufacturer recommended Amplicons from the respective plate are removed from the thermal cycler uncovered and pipetted into the formamide master mix plate with an 8 channel pipette The amplicon formamide size standard plate is covered with a 3500 septa and denatured for a few minutes followed by an ice block cooling for a few minutes The denatured plate is installed in the 3500 the 3500 analyzer allows two plates to be insta
118. tudy Analysis Threshold 150 RFU Stutter Study Adopted max stutter values from a comparison between WSCL and Applied Biosystems values 2 Developmental Validation Applied Biosystems 2010 AB 3500 and 3500xI Genetic Analyzer Specification Sheet 62 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice Applied Biosystems 2011 AB AmpFf STR Identifiler Plus PCR Amplification Kit User Guide Developmental Validation section 5 page 70 user guide rev Applied Biosystems 2010 Genemapper ID X Software Version 1 2 User Bulletin 3 Internal Validation 3 1 Known and non probative evidence samples The method must be evaluated and tested using known samples and when possible authentic case samples otherwise simulated case samples should be used DNA profiles obtained from questioned items should be compared to those from reference samples When previous typing results are available consistency as to the inclusion or exclusion of suspects or victims within the limits of the respective assays should be assessed Concordance Study Peak Height Ratio Study Stutter Study Qualifying Test 3 2 Reproducibility and precision The laboratory must document the reproducibility and
119. ults and use the manufacturer s 35 This document is a research report submitted to the U S Department of Justice This report has not been published by the Department Opinions or points of view expressed are those of the author s and do not necessarily reflect the official position or policies of the U S Department of Justice applicable guides e g 3500 User Guide Biology Exhibit 59 01 to perform spatial calibration troubleshooting gt Perform a spectral calibration for all dye sets currently used on the instrument Spectral Calibration A spectral calibration creates a de convolution matrix that compensates for dye overlap reduces raw data from the instrument in the multi dye data stored in each sample file A spectral calibration may be necessary if there is a decrease in spectral separation pull up and or pull down peaks in the raw or analyzed data or if the capillary array has been changed Spectral calibration for dye set G5 e g Identifiler Plus gt Complete a spatial calibration section 5 2 2 if not previously performed Verify consumables are not expired and adequate injections remain for consumables Ensure buffer levels are at the fill lines Pre heat the oven to 60 C Start Pre Heat button on main dashboard a Applied Biosystems recommends pre heating the oven for at least 30 minutes before a run is started if the instrument is cold Pre heating mitigates first run migration e
120. use of this kit and the analytical procedure for amplifying DNA for genetic typing using STR technology 2 Extraction Methods and Controls A Extraction Methods N A direct amplification procedure B Controls Reference Databasing Technical Manual 2 2 2 Amplification Controls 3 Procedure QlAgility Based PCR Setup A Turn on the computer for the QIAgility B Turn on the QIAgility instrument C Activate the QIAgility software D Open the appropriate protocol for Identifiler Plus with direct amplification from the Protocols folder on the desktop i Validated protocols include the ID Plus 90s and ID Plus 90s small mix protocols Most routine database runs will utilize the D Plus 90s protocol as the D Plus 90s small mix protocol is for partially full plates with 45 or fewer samples A smaller master mix tube is utilized in the small mix protocol otherwise the protocols are identical E Check the QlAgility deck setup to ensure it reflects the virtual deck setup in the software e g pipette tip types placements quantities sample blocks F Load a 5ml master mix tube into position A of the Mix Plate G Load the Identifiler Plus Reaction Mix into position A of the reagent plate and the lIdentifiler Plus Primer Set into Position B of the reagent plate 29 r This document is a research report submitted to the U S Department of Justice This report has not been published by the Department

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