Archer FAQs PDF
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1. 4 Archer Frequently Asked Questions MKT 0001 Rev D a J ct AR F ARCHER N wer i e Negative control the Human Blood Leukocyte Total RNA Ambion R1234148 10 Q Are there any commercially available positive or negative controls for the Archer FusionPlex FGFR fusion detection assay A Yes we recommend the following for positive and negative controls e Positive control the Negative BCR ABL p210 amp p190 that is part of the psogen BCR ABL Mbcr Control Kit QIAGEN 670191 is positive for the FGFR1 FGFR10P2 fusion e Negative control the Human Normal Thyroid Total RNA Ambion R1234265 50 Sample Multiplexing Adapter Ligation amp First Second PCR Steps Q What are the part numbers for the Molecular Barcode Adapters used in the Archer FusionPlex Assays Please click on the links in the list below for more details regarding the MBCs or http archerdx com mbc adapters Catalog Molecular Barcode Adapter MBC Description SA0027 Archer Barcode Adapters 1 8 for lon Torrent Platform SA0028 Archer Barcode Adapters 9 16 for lon Torrent Platform 540029 Archer Barcode Adapters 17 24 for lon Torrent Platform SAQ03Q Archer Barcode Adapters 25 32 for lon Torrent Platform SA0031 Archer Barcode Adapters 33 40 for lon Torrent Platform SA0032 Archer Barcode Adapters 41 48 for lon Torrent Platform SA0040 Archer MBC Adapters A1 A8 for Illumina SA2. 19093 instead of harsh chemical treatments such as Xylene e Do not treat the extracted total nucleic acid with DNase Doing so may critically reduce the quality of RNA in the sample e Follow the RNA extraction protocol that DOES NOT include small RNAs lf DNAse treated my FFPE sample during the extraction process will the assay function correctly DNAse treatment can reduce the quality of the RNA in the sample When using FFPE samples total nucleic acid is the recommended starting material z7 OQ Q What is the minimum amount of starting material for the assay A The minimum recommended input for the assay is 20ng total nucleic acid The amount of input material can be adjusted according to the quality of the RNA sample When using less than 10ng input material the number of PCR cycles for First and Second PCR may need to be increased Note that reduced sample input may adversely affect read diversity Please refer to the following Instructions for Use 2 Archer Frequently Asked Questions MKT 0001 Rev D N ARCHER a AK0024 8 Archer Universal RNA Reagent Kit for Illumina Platform AK0040 8 Archer Universal RNA Reagent Kit v2 for Illumina 8 AKO025 8 Archer Universal RNA Reagent Kit for lon Torrent Platform AKO042 8 Archer Universal RNA Reagent Kit v2 for lon Torrent 8 Q What is the recommended maximum input amount for the kits A If higher library complexity is desired the assay can tole
3. 300 000 500 00 FusionPlex Heme Panel 132 1 000 000 5 000 000 FusionPlex Sarcoma Panel 134 1 000 000 5 000 000 300 000 500 00 FusionPlex NTRK Panel 17 300 000 500 00 FusionPlex FGFR Panel 17 H of Recommended of Archer lon Torrent Panel Targets Assay samples 318 Chip FusionPlex ALK RET ROS1 Panel v2 29 3 FusionPlex Heme Panel 132 2 3 FusionPlex Sarcoma Panel 134 Das FusionPlex NTRK Panel 17 7 10 FusionPlex FGFR Panel 17 Pat Does Agencourt AMPure purification affect the complexity of my prepared libraries No the AMPure XP beads are designed to efficiently remove smaller molecules such as unincorporated dNTPs primers primer dimers salts and other contaminants Will AMPure XP bead carryover impact any downstream steps Remove all beads if possible However a very minimal amount of AMPure beads has no noticeable impact on the assay Why was a new polymerase Phoenix Taq chosen for PCR 1 and 2 ArcherDX is committed to continuously improving our product line for both the chemistry and software The Phoenix Taq enzyme results in less mis priming leading to more reads from the correct targets or less false positive results Why does the Universal RNA Kit v1 have a ramp rate from 98 C to 68 C while the Universal RNA Kit v2 have aramp rate from 95 C to 65 C The ramp rates were optimized for each version of the kit The RNA Kit v1 and v2 have different ramp rates because different enzymes are used for PCR The RNA Kit v1 uses VeraSe
4. background DNA will not interfere with RNA quality assessment A Ct cutoff is experimentally determined for all RNA samples above which samples will not pass QC on Archer Analysis Q What is the minimum RNA fragment size for starting material A The assay requires RNA fragments to be a minimum of 60 to 70 bases in length The Universal RNA Reagent kit v2 AKO040 8 AK0042 8 contains RNA QC primers that will amplify a 113 bp amplicon of the VCP gene After first strand cDNA synthesis the sample can be tested by qPCR to determine whether to continue with the assay Please refer to the Instructions for Use for more information 3 Archer Frequently Asked Questions MKT Q001 Rev D N ARCHER a AK0024 8 Archer Universal RNA Reagent Kit for Illumina Platform AK0040 8 Archer Universal RNA Reagent Kit v2 for Illumina 8 AKO025 8 Archer Universal RNA Reagent Kit for lon Torrent Platform AK0042 8 Archer Universal RNA Reagent Kit v2 for lon Torrent 8 Kit Storage amp General Protocol Q What are the recommended kit storage conditions A Store all components of the Universal RNA Reagent kits at 4 C The liquid gene specific primers GSP1s and GSP2s should be stored at 20 C Q The Archer RNA Universal Reagent kit was accidentally stored at 20 C Can it still be used A Storage at 4 C is ideal however accidental storage of the kit at 20 C will not adversely affect kit performance Bring to room
5. installed Archer Analysis on my virtual machine but it requires me to open my browser to run it Am online A No you are not The virtual machine generates a local IP address that you copy into your browser however you are only running on the local machine not online In the example below the virtual machine is running on the IP address 192 168 56 101 which is internal to the organization and does not exist outside the firewall of the organization eee Home ArcherDx x lt e 192 168 56 101 home a Settings ARCHER ANAYAM Gi AE AT CADUA ANKA amp WIMA GA AA Ue A 3 NEON KA A a A A N CATLA ASA K LAM CACAL AK Running and Recent Jobs Job Job Details Q How do know when my run is complete A Open the Archer Analysis home page on your virtual machine browser and check the Job Status beside your run If the run is complete you can click on the detailed summary of the run to see sample data As shown in the example below 1992 ARR Demo RNA Fusion RNA COMPLETED OKAY FusionPlex ARR Illumina paired ee SNP InDel Panel V2 Started February 18 2015 03 43AM ae Deduplication molbar Completed February 18 2015 11 26AM E7 2 12 Archer Frequently Asked Questions MKT 0001 Rev D ARCHER i Q What are the meanings of the symbols on the summary and evidence pages A JH3350 SLC34A2 ROS1 31 LO01 R1 001 Strong Evidence Fusions Detailed Summary Processi
6. to your target of interest You do however need to specify the exon of interest for each target as well as the direction of the fusion partner from the target exon 3 or 5 Note that mid exon breakpoint primer design is not supported by Assay Designer at this time If you want to detect mid exon breakpoint fusions contact technical support at tech archerdx com Archer Frequently Asked Questions MKT Q001 Rev D ARCHER For further assistance please contact Archer Technical Support Email tech archerdx com Phone 877 771 1093 or 303 357 9001 Limitations of Use For Research Use Only Not for use in diagnostic procedures This product was developed manufactured and sold for in vitro use only The product is not suitable for administration to humans or animals SDS sheets relevant to this product are available upon request 2015 ArcherDX Inc All rights reserved Archer and Archer FusionPlex are trademarks of ArcherDx Inc Illumina HiSeqg NextSeq_ MiSeg and Nextera are registered trademarks of Illumina Inc Agencourt AMPure and FormaPure are registered trademarks of Agencourt Biosciences Corporation a Beckman Coulter company Life Technologies Ambion lon Torrent SYBR Applied Biosystems GeneAmp Veriti and DynaMag are trademarks of Thermo Fisher Scientific Inc Oubit is a registered trademark of Molecular Probes Inc KAPA Biosystems is a registered tra
7. 0041 Archer MBC Adapters A9 A16 for Illumina SA0042 Archer MBC Adapters A17 A 24 for Illumina SA0043 Archer MBC Adapters A25 A32 for Illumina SA0044 Archer MBC Adapters A33 A40 for Illumina SA0045 Archer MBC Adapters A41 A48 for Illumina AKO016 48 Archer MBC Adapters Set B for Illumina AKO017 48 Archer MBC Adapters Set C for Illumina Q What is the best way to use indexes for multiplexing samples A See the Sample Multiplexing section of the appropriate Archer Universal RNA Reagent Kit Instructions for Use for details Select the document below for all relevant information AKO024 8 Archer Universal RNA Reagent Kit for IIlumina Platform AK0040 8 Archer Universal RNA Reagent Kit v2 for Illumina 8 AKO025 8 Archer Universal RNA Reagent Kit for lon Torrent Platform AKO042 8 Archer Universal RNA Reagent Kit v2 for lon Torrent 8 D Archer Frequently Asked Questions MKT Q001 Rev D F ARCHER SS en i PO 4 o gt AR How many samples can be multiplexed together The level of multiplexing depends on the number of targets and the number of reads generated by the instrument per run This will vary for each catalog panel as well as custom panels Custom fusion detection assays will need to be optimized to balance the number of reads needed against the level of multiplexing of Recommended of Archer Illumina Panel Targets Assay Reads FusionPlex ALK RET ROS1 Panel v2 29
8. 8 98 6 97 39 A Archer Analysis utilizes BWA and Bowtie 2 for mapping Any read that has an Alignment Score less than 30 is removed from consideration Reads with a mapping quality less than 30 are either multiple mapping reads or contain many low quality bases low FASTQ base quality For the exact calculation of the mapping quality and the factors involved please refer to the BWA and Bowtie 2 documentation Note that this default value can be adjusted by the user in the Analysis Settings area of Archer Analysis BWA http bio bwa sourceforge net bwa shtml Bowtie http bowtie bio sourceforge net index shtm Q What are the criteria for the QC Result A A sample will pass the QC filter if the number of unique de duplicated RNA reads per target Gene Specific Primer 2 GSP2 is at least 200 This has proven to be a reasonable criterion for the success of the assay However users are encouraged to evaluate the results themselves and set their own cutoff Q What are the criteria for fusion candidates A Fusion candidates are required to have a minimum of 5 unique de duplicated break point spanning reads that support the gene fusion This is the default value however users can adjust this default value in the Analysis Settings area of Archer Analysis They must also comprise at least 10 of the total reads for that GSP2 not be intron exon fusions and not show any similarity to each other to be considered a Strong Fusion cand
9. DNA RNA 80234 1 Archer Frequently Asked Questions MKT 0001 Rev D ARCHER a R ee lt DA Fresh frozen FF Any total RNA 10mM Tris or Qubit RNA HS Agilent RNA 6000 tissue cell lines and extraction kit nuclease free Assay Kit Life Nano RNA blood water Technologies Bioanalyzer Assay No EDTA 032852 5067 1511 Q Do you have any suggestions for using the Agencourt FormaPure kit A33342 when performing nucleic acid extraction of FFPE samples A We do have modifications to the published instructions e DO NOT treat the extracted total nucleic acid with DNase Doing so will critically reduce the quality of RNA in the sample e Be sure to follow the total nucleic acid extraction workflow in the Agencourt protocol which does not include the DNase treatment e Use heat blocks versus water baths throughout the protocol e Perform an overnight digestion of FFPE samples at 55 C for 16 hours at Step 5 of the Agencourt protocol e Elute the sample in 40uL nuclease free water at Step 23 This is the minimum elution volume see our modified FormaPure FFPE Sample Prep ha demonstration at http archerdx com agencourt formapure ffpe extraction suggestions html FormaPure FFPE Sample Prep Q Do you have any suggestions for using the QIAGEN All Prep DNA RNA FFPE kit cat no 80234 when performing nucleic acid extraction of FFPE samples A We do have some suggestions e Use QIAGEN Deparaffinization Solution cat no
10. Instructions for Use Archer Frequently Asked Questions For products including Catalog Description AK0024 8 Archer Universal RNA Reagent Kit v1 for Illumina Platform AK0040 8 Archer Universal RNA Reagent Kit v2 for Illumina 8 AK0025 8 Archer Universal RNA Reagent Kit v1 for lon Torrent Platform AKO042 8 Archer Universal RNA Reagent Kit v2 for lon Torrent 8 AKO028 8 Archer FusionPlex ALK RET ROS1 Panel v2 AK0029 8 Archer FusionPlex Heme Panel AKO030 8 Archer FusionPlex FGFR Panel AK0031 8 Archer FusionPlex NTRK Panel AK0032 8 Archer FusionPlex Sarcoma Panel Variable Archer FusionPlex Custom Q Which control genes are used in the Archer FusionPlex Assays A The control genes for the assays are CHMP2A RABA7A GPI and VCP These are housekeeping genes required for the maintenance of basic cellular function Input Nucleic Acid Concentration and Purification Q What types of starting material are recommended for the kits and how should they be processed A Recommended Recommended Quantification Recommended Starting Material Extraction Method Elution Buffer Method Quality Check Step Formalin fixed Agencourt 10mM Tris or Qubit RNA HS Agilent RNA 6000 paraffin embedded FormaPure Total nuclease free Assay Kit Nano RNA FFPE tissue Nucleic Acid water Life Bioanalyzer Assay Extraction No EDTA Technologies COG esse A33342 or 032852 QIAGEN AllPrep
11. ample will not show PASS in the QC section However depending on your samples the data may be fine especially if the number is close to 200 Any positives detected are real if they meet all the other cutoffs The minimum number of unique reads is set to ensure there are no false negatives This is the reason the cutoff should be set by each lab during validation 13 Archer Frequently Asked Questions MKT Q001 Rev D he a i r ARCHER Se TO 14 A d EEEN D What is a molecular barcode MBC A molecular barcode is a unique 8 bp sequence added to the molecule to identify unique fragments and de duplicate the sequencing reads from a sample This along with the random start sites helps identify and remove PCR duplicates Learn more about the value of MBCs for accurate data analysis Watch the videos at http archerdx com mbc adapters TITTY TT Tt tees UT Molecular Barcode DOCO Identification of unique fragments OK OK OK OK What percent of my total fragments can expect to have a molecular barcode This percentage varies with library quality For high quality libraries the fragments with molecular barcodes should be 80 90 of the total fragments What if have a very low number of fragments with molecular barcodes This reduces the total number of fragments that are analyzed and may cause the sample to not pass QC but does not necessarily mean the run or results are bad This can potentially be
12. cherdx com videos How much does the software cost Nothing Customers using any of the Archer panels can create an account and download the software for free Visit archerdx com analysis for complete information regarding Archer Analysis Do need to download the virtual machine or command line version of the software to try it Users are not required to use either of these approaches ArcherDX provides a free web based version of Archer Analysis Data on this server is not guaranteed to be secure or private and is primarily meant for demonstration purposes Customers may also access a set of demo data using the link and login information below http archerdx com demo data User Name example example com Password password123 NOTE Data loaded on this server will be removed after 30 days What are the requirements for the virtual machine and command line versions of Archer Analysis Users must have a virtualization application such as Virtual Box or VMWare installed on their system first The system must have at least 12GB free RAM total system RAM 16GB and 200GB available storage Download the analysis package from http archerdx com analysis This package contains all of the analysis functionality in a single package that does not require any extra software other than Virtual Archer Frequently Asked Questions MKT 0001 Rev D Eo ARCHER ae i an S gt AW Machine software The command line versi
13. demark of KAPA Biosystems Inc Bio Rad T100 C1000F S1000f and iTaq are trademarks or registered trademarks of Bio Rad Laboratories Inc Bioanalyzer is a registered trademark of Agilent Technologies Inc RNase Away is a registered trademark of Molecular Bio Products Inc jpsogen and QIAGEN are registered trademarks of QIAGEN GmbH For more information please visit http www archerdx com ArcherDX Inc 2477 55 Street Suite 202 A R C H E R Boulder CO 80301 303 357 9001 _7 www archerdx com 20 Archer Frequently Asked Questions MKT Q001 Rev D
14. ene or fusion as well as which exons participate in the known fusion and links to PubMed and other public resources This is useful for researching fusions that you are unfamiliar with or when designing your own custom assay Q Can I add new fusions to Quiver A Currently you cannot but we are working on adding that function For now if you find a novel fusion contact tech support at tech archerdx com 17 Archer Frequently Asked Questions MKT 0001 Rev D N ARCHER ai Archer Custom Design Tools Assay Designer Q A 18 What is Archer Assay Designer Assay designer is our free online tool used to create your own custom assays It can be found here http assay archerdx com assay How do create a custom fusion panel A custom fusion panel can be made at http archerdx com assay designer Exons from genes can be selected and added to the custom fusion panel It is also possible to upload a csv file with the appropriate information Once the design has been submitted a representative from ArcherDX will contact you to make sure the design is correct The Quiver Fusion database http archerdx com quiver can also be used to find known fusions that can then be added to the custom design Why do I have less than 100 coverage of my target Assay Designer might fail to design a primer to a target region for one or more of the following reasons 1 Target region has high similarity to other regions in t
15. era chemistry Please note that our libraries are dual indexed and should be sequenced using 2x150bp PE reads and 2x8bp index reads For Nextera you will need to add BP13 primers included in the kit Refer to the Instructions for Use below for guidance Archer Frequently Asked Questions MKT Q001 Rev D N ARCHER a AK0024 8 Archer Universal RNA Reagent Kit for Illumina Platform AK0040 8 Archer Universal RNA Reagent Kit v2 for Illumina 8 AK0025 8 Archer Universal RNA Reagent Kit for lon Torrent Platform AKO042 8 Archer Universal RNA Reagent Kit v2 for lon Torrent 8 How much PhiX should add to my library for the Illumina instrument platforms Add 10 PhiX to the library to serve as a control on the Miseg and add 30 PhiX on the NextSea What cluster density can be expected on the MiSeq This will vary by kit version We recommend using the MiSeg Reagents Kit V2 300 cycle MS 102 2002 The typical cluster density with the recommended loading concentration of 10pM will be approximately gt 700 k mm Archer Analysis Software Installation Q A z7 O 10 Where can find the instructions to install and run Archer Analysis The complete Installation Guide and Instructions for Use can be downloaded from the following link http archerdx com assets documents Archer Analysis Manual pdf Additionally informational videos and links to other supporting materials can be found at http ar
16. he genome which would result in off targeting 2 GC content in target region is too high or too low 3 There are known mutations present in the primer site Can adjust the primer search parameters to recover missed targets No The primer search setting in Assay Designer are fixed so that primers are optimal and likely to be fully functional with our assay If you need assistance recovering important targets contact technical support for assistance at tech archerdx com Are my primers tested and balanced before order them No Primers are designed in silico and ordered directly through Assay Designer They are not tested in any way prior to shipping They will arrive in an equimolar pool For best results it is recommended that you test the primer pool on DNA before use for fusion detection Testing the primers on DNA will allow you to confirm that all primers are fully functional without the added complication of expression levels associated with target amplification of RNA If you are interested in having ArcherDx functionally test your primers and balance your primer pools contact technical support at tech archerdx com What is the minimum number of targets can include in an assay There is no current minimum However the standard 8 controls included in our catalog panels will be included in any design Archer Frequently Asked Questions MKT Q001 Rev D ARCHER i OQ O oO 19 What is the maximum n
17. idate Fusions that are found in the Quiver database are always marked as Strong Fusion candidates regardless of any of the other filters 2 Settingsy 4Admin Help ARCHER An S IS aeaa or gt Analysis Settings eee PERFORM R amp D Option ptions ANALYSIS ABOUT ARCHER LOG OUT Manage Custom Targets Manage Targeted Mutations Archer Analysis Settings MIN_READS_FOR_VALID_ FUSION 5 Archer Analysis Troubleshooting Q My job status says COMPLETE ERROR What should do 15 Archer Frequently Asked Questions MKT Q001 Rev D ARCHER N i ee AR 2066 OSU all 6 RNA Fusion COMPLETED ERROR FusionPlex Sarcoma Panel lon Torrent Submitted by tdeboer enzymatics com Deduplication molbar V1 demultiplexed Started March 05 2015 12 30PM Completed March 05 2015 05 11PM E View All Analysis Logs A Make sure that you have 1 Selected the correct de duplication method 2 Selected the correct platform option Illumina paired end is the default If all these are correct then contact Archer technical support at tech archerdx com or 877 771 1093 Consult the Analysis Logs by selecting the View Analysis Logs option B to view the log files to obtain clues about why the error was produced If possible send the log files to ArcherDX technical Support to help in the error analysis Q chose the correct parameters but my sample still failed QC now what Strong Evidence Fusio
18. iversal RNA Reagent Kit v1 and 15 to 100nM for the Universal RNA Reagent Kit v2 Do not use libraries with concentrations below 2nM Why is my final library concentration low Reasons for low final library concentrations include 1 The source of the nucleic acid the purification method the chemical the nucleic acid is suspended in and the starting amount of nucleic acid can contribute to low library concentration Extract RNA from FFPE tissue with one of our recommended methods Make sure RNA input quantity was measured using the HS RNA Qubit assay and sufficient starting material was used Additionally the sample should pass the PreSeg VCP qPCR QC step which can be performed after first strand cDNA synthesis to determine whether to continue with the assay or repeat the nucleic acid extraction and start over Refer to the protocol link to the protocol for more information on the PreSeg RNA QC step 2 Incorrect PCR cycling can lead to low library yields Ensure that the cycling temperatures cycle numbers and ramp rates are accurate Check the instrument to make sure it is calibrated and in good working order My template negative control produced sequenceable library Is this normal Yes a template negative control will fail the QC filter in Archer Analysis and thus will not produce meaningful results Are MiSeq libraries compatible with the HiSeq and NextSeq instruments Yes as long as they are run on a paired end flowcell using Next
19. load files to Archer Analysis We recommend that you put all of the FASTQ files to be uploaded into a single location Note that all of the files for a single job must be uploaded at the same time Make sure to select and upload both the read 1 and the read 2 FASTQ files for each sample when working with the Illumina paired end reads libraries How long will it take to upload my files This will depends on your internet speed and file size You can check your upload speed at http speedtest net and calculate your upload time by dividing the sum of your file size by your upload speed 50134 216 103 Camcast Cable Hosted by FORETHOUGHT net In this example the speed is 44 38 megabytes second You would divide your file size by this number Archer Frequently Asked Questions MKT Q001 Rev D ARCHER i Q How long does Archer Analysis take to analyze data A The analysis of a single set of FASTQ files 250 000 reads or 50 100MB data should take only a few minutes Larger runs of multiple samples can take a few hours depending on the complexity and amount of reads By default the VM runs each sample serially but the system can be set up to process multiple samples at the same time Consult the Instructions for Use about how to set up Archer Analysis for parallel processing of samples The Archer Analysis Instructions for Use are located at http archerdx com assets documents Archer Analysis Manual pdf Q
20. ng Log VEP Variant Overview Visualize Sample Data SLCE34A2 ROS1 SLC34A2 gt ROS1S BRWD1 SLC34A2 FUSION QC PASS VARIATION QC PASS Novel Isoforms Variants Found Means that the fusion passed all QC Metrics Means that the fusion is a known fusion according to the Archer Quiver database iti Means that the percent fusion reads for the GSP2 detecting that fusion is below the set threshold Means that the fusion partners show sequence similarity to each other and could therefore be a mispriming event Means that the fusion is an exon intron fusion Most fusions are exon exon fusion Q How is my data kept confidential A To ensure sample confidentiality be sure to run Archer Analysis on a local machine and not the web based option This keeps your data on your local system not the web Archer Analysis Expected Metrics Q How many unique fragments reads do need per sample A The number of fragments needed depends on the number of targets in your assay and the quality of your sample The default cutoff is 200 unique fragments per target GSP2 You can calculate the number of unique RNA reads on target you need to pass our standard QC cutoff by multiplying the number of targets in your panel by 200 This cutoff should be evaluated during validation and set appropriately for each lab Q What if do not have 200 unique RNA reads per GSP2 A Unless you have updated your analysis settings your s
21. ns No Strong Evidence Fusions Detected A This does not necessarily mean that your data is bad Click on Detailed Summary to get more information about why it failed and decide if you want to accept the data change parameters or re run the sample Read about what is acceptable data in these other FAQs 1 What is the most common reason runs fail QC 2 How do know if my fusion is real Q What are the most common reasons runs fail QC A Run QC is most affected by starting library quality fragment uniqueness and coverage You will see the effects of these factors on Average Unique Fragments per GSP2 because it takes many metrics into account This is found under the Read Statistics tab in the 3 set of boxes The default setting is 200 Average Unique Fragments per GSP2 for RNA reads You might want to change the 200 cutoff based on your validation results DNA RNA Statistics JH3343_EML4 ALK_ 24 LOO1_R1_001 DNA Reads RNA Reads Ambiguous Reads All Fragments 67 586 0 17 1 195 584 0 494 132 767 0 335 Unique Fragments 3 300 0 174 8 355 0 44 1 7 293 0 38 5 Average Unique Fragments per GSP2 99 79 286 76 230 97 16 Archer Frequently Asked Questions MKT 0001 Rev D ARCHER i Q How do I know if my fusion is real A Archer has provided information to guide the detection of fusions and variations However every sample is different Results must be interpre
22. on Kit A Using the Qubit instrument is not recommended for the final library concentration We cannot guarantee consistent loading concentrations with the Qubit because a size selection of the final library is not performed Therefore we recommend using the appropriate KAPA Biosystems Library Quantification Kit for accurate quantification of sequenceable molecules Other commercially available gPCR based library quantification kits can also be utilized Please refer to the Instructions for Use below for guidance 8 Archer Frequently Asked Questions MKT Q001 Rev D N ARCHER a z7 O AK0024 8 Archer Universal RNA Reagent Kit for Illumina Platform AK0040 8 Archer Universal RNA Reagent Kit v2 for Illumina 8 AKO025 8 Archer Universal RNA Reagent Kit for lon Torrent Platform AK0042 8 Archer Universal RNA Reagent Kit v2 for lon Torrent 8 What is the average amplicon length for a FusionPlex assay The expected average size for amplicons will range between 150 and 400 base pairs as viewed on a Bioanalyzer trace However you should assume an average fragment length of 250 base pairs when using the KAPA Biosystems Library Quantification Kit for qPCR Our recommended dilutions and MiSeq and PGM input amounts are all based on an assumed average fragment length of 250 base pairs What is the expected concentration of my final library Final library concentration should range between 50 and 500nM for the Un
23. on is provided for customers that would like to integrate Archer Analysis with their own analysis pipeline What virtualization software can be used to run the virtual machine version of Archer Analysis We have extensively tested with Oracle Virtual Box for workstations and VMware vSphere for larger installations but other virtualization software should work equally well Oracle VirtualBox can be downloaded using this link httos www virtualbox org How much free memory should my machine have to run the virtual machine version of Archer Analysis The system with the virtual machine will need at least 12 GB of free memory RAM This will allow a user to run a single sample at a time More memory 12G more per sample will allow users to run multiple Samples at a time What web browser s are supported with Archer Analysis While most current browsers are supported we recommend using Chrome or FireFox Note that Internet Older versions of Internet Explorer do not support multiple selections of FASTQ files in the file dialog box however most recent versions of Internet Explorer on work correctly Can keep using the old version of Archer Analysis after you release a new version Yes this is one of the benefits of using virtual machines You can have a virtual machine with each version of the software to allow for continuity and ease of validation Archer Analysis Getting Started Q A Q A 11 What is the best way to up
24. ooling ramp rate Q Is the modified ramp rate in either the 68 C or 65 C PCR step meant for ramping toward that temperature or away from it A Ramping towards that temperature Therefore the ramp rate should be set at either 2 3 C second moving down from 98 C to 68 C in Universal RNA Reagent Kit v1 AKO024 8 AKO025 8 or at 0 28 C second moving down from 95 C to 65 C in the Universal RNA Reagent Kit v2 AKO040 8 AKO042 8 Ramp rates Universal RNA Reagent Kit v1 PCR 1 conditions heated lid Incubation temperature Incubation time Volume 20uL cycles 7 Archer Frequently Asked Questions MKT 0001 Rev D N ARCHER a Adjustable PCR 2 conditions heated lid Volume 20uL Incubation temperature Incubation time of cycles as 870 D sn m N 68 C ramp rate of 2 3 s bS 72 C 3 min 1 ite ie jacana Adjustable Ramp rates Universal RNA Reagent Kit v2 PCR 1 conditions heated lid Volume 20uL Incubation temperature Incubation time cycles 7 Sloe 3 min 1 a T ae 65 C ramp rate of 0 28 C s 60 s ee ea a e e D a a a Adjustable PCR 2 conditions heated lid Volume 20uL Incubation temperature Incubation time of cycles La ne So 95 C BOS ne 7 65 C ramp rate of 0 28 C s 60 s mig a 4 C HOLD 1 Adjustable Quantify Library and Sequence Q Can a Qubit instrument be used to measure the concentrations of the final libraries instead of a KAPA Biosystems Library Quantificati
25. overcome by performing deeper sequencing What percent of my fragments with a molecular barcode can expect to be unique For good libraries with the correct amount of starting material up to 20 of the fragments with molecular barcodes should be unique fragments For FFPE samples the percentage of unique fragments can drop to 1 10 However this is not a firm cutoff and varies with library quality and panel What does the On Target percent mean On Target percent is the percentage of library fragments that include the entire GSP2 which requires that the fragment be long enough to reach the GSP2 and be of high enough quality at the end of that read to identify all bases in the GSP2 Under the Read Statistics tab of the Detailed Summary page how do you define a DNA vs RNA vs ambiguous read e ADNAread is a read that spans an intron exon junction e An RNA read is a read that spans an exon exon junction e An ambiguous read is a read that does not span any junction and therefore cannot be identified as DNA or RNA Archer Frequently Asked Questions MKT Q001 Rev D ARCHER Q How is the mapped fragment number calculated Under the Read Statistics tab of the Detailed Summary page Read Statistics ROS 100ng_S 81_L001_R1_001 combined Total Fragments Mapped Pass Alignment Score Filter On Target All FragmentsO 728 933 727 366 99 79 99 8 99 5 Unique FragmentsO 84 621 83 419 98 5
26. q Tag for PCR and the RNA Kit v2 Archer Frequently Asked Questions MKT 0001 Rev D N ARCHER a o A eS DD uses Phoenix Taq for PCR The modified ramp rate in the v2 kit is vital to the assay s function due to the complex nature of the Anchored Multiplexed PCR reaction It is important to verify that this ramp rate is set correctly before starting the PCR program by consulting the instrument s user manual or contacting the manufacturer Please see the table below for v2 kit ramp rates for specific thermal cyclers Company Applied Biosystems Life Technologies Bio Rad Eppendorf Thermal Cycler Model GeneAmp PCR System 9700 Veriti TOOR C1000 S1000 Mastercycler Pro Mastercycler Pro 5 Mastercycler Pro 384 Block Type 60 wells 96 Aluminum 96 Silver Gold 2 x 96 3 x 384 Standard 96 Fast 96 60 wells 96 wells 96 wells 96 wells deep block 2 x 48 block 384 well block 96 wells 96 wells deep block 384 well block 96 Aluminum 96 Silver Aluminum Maximum Block Ramp Rate 2 s ILS 23 ZG T6 1 8 3 9 9 0 3 9 4 0 50 9 0 4 0 3 0 6 0 4 5 4 0 3 0 Notes set ramp under the denaturing step of the amplification cycle can only be set at 0 2 C or 0 3 C s Default to 0 2 C s in this case Block ramp rate heating ramp rate cooling ramp rate Block ramp rate heating ramp rate cooling ramp rate Block ramp rate heating ramp rate c
27. rate up to 250ng total nucleic acid too much input will decrease final library yield Q Can Tris EDTA buffer be used instead of 10mM Tris HCl pH 8 0 buffer to elute and store my samples A Do not use buffers with EDTA Use nuclease free water or 10mM Tris HCl pH 8 for sample storage Q Can genomic DNA be used with this protocol A Not at the present time Archer Universal RNA Reagent kits are designed for total nucleic acid or RNA as the Starting material Q What should do if my input sample is too dilute to run the Archer assay A Repeat the nucleic acid isolation to increase quantity of starting material available for the assay Q How can check the quantity and quality of my RNA sample e Determine the sample concentration on the Qubit instrument using the HS RNA Assay Kit Life Technologies 032852 e Visualize the size distribution and RNA integrity on an Agilent Bioanalyzer RNA 6000 Nano Kit 5067 1511 e Universal RNA Kits version 2 AK0040 8 AKO042 8 come with PreSeq qPCR QC to assess RNA quality and help predict whether or not there is sufficient good quality RNA to pass QC PreSeg can also be purchased separately from the RNA assay if you would like to screen all RNA samples prior to proceeding through library preparation Q How does PreSeg work A PreSeg primers target a 113 bp region of VCP one of the housekeeping control genes targeted in the Archer assay The 113 bp region spans an exon exon junction so
28. ted by each lab and take into consideration any information know about their particular samples Read statistics and the visualization tools can assist in making proper calls Some things to consider include 1 Does the fusion appear in the strong evidence tab 2 Are there several reads supporting the fusion call 5 is the MINIMUM number of unique reads to call a fusion Does the fusion have a large percent of the reads as compared to the wild type transcript Is the expression level of the target gene normally high or low How does the expression level of the target in the sample compare to normal Is the fusion known in the literature a m D a When visualizing the data are there any insertions or deletions present in more than a few of the reads near the breakpoint This could indicate a mapping error 8 When visualizing the data do all of the reads have the same start or stop point If so they could be duplicates Learn more about visualizing your fusions and reads in JBrowse Watch the video at wee http archerdx com support fags archer analysis troubleshooting how do I know if my fusion is real Quiver Q What is the Quiver Fusion Database A The Quiver Fusion Database is a compilation of known fusions from several public databases Visit http archerdx com quiver to learn more about it Q What can I use Quiver for A Quiver provides information on which of our panels include a particular g
29. temperature prior to use Q The kit was accidentally left out overnight on the bench top Can it still be used A Yes you can use the kit if it has been left out overnight at room temperature However the pouches must have remained sealed with the desiccant packs inside Q Are there stopping points in the protocol A There are four stopping points in the workflow 1 After Second Strand cDNA Synthesis 2 After Adapter Ligation 3 After First PCR 4 After Second PCR Q How long does the library preparation protocol take to perform without stopping A The library preparation protocol takes approximately 7 9 hours of total time depending on the version of the kit used and 2 hours of hands on time to process 8 samples Q Are there any commercially available positive or negative controls for the Archer FusionPlex ALK RET ROS1 v2 fusion detection assay A Yes we recommend the following for positive and negative controls e Positive control an ROS1 positive RNA sample such as the HCC 78 cell line from Creative Bioarray CSC CO569 e Negative control the Ambion Human Lung Total RNA Life Technologies AM7968 Q Are there any commercially available positive or negative controls for the Archer FusionPlex Heme fusion detection assay A Yes we recommend the following for positive and negative controls e Positive control the High BCR ABL p210 control that is part of the jpsogen BCR ABL Mber Control Kit QIAGEN 670191
30. umber of targets can include in a panel There is no specific maximum number of targets for Assay Designer However we will flag panel designs that we do not believe in si co design is sufficient to meet your expectations or ours In this instance we will be in contact with you to discuss specifics and how you would best like to proceed with procuring a custom panel How long will it take to receive my custom primer order after submitting my order After the design has been approved and submitted for order you can expect to receive your equimolar primer pools in 6 8 weeks ALK_E_r_020_4_R_0_GSP2 Why do my primers have to be reviewed before can place my order To provide you with the best chance of creating a successful primer design for your assay a member or our team will carefully review each primer in your design and remove any primers expected to perform poorly In some cases we might recommend that the custom panel be redesigned by our in house R amp D team to ensure that they meet both your and our expectations Do need to specify a breakpoint in order to detect fusion Assay Designer assumes that all breakpoints are at the specified end of the exon 3 or 5 If you want to detect mid exon breakpoint fusions contact technical support at tech archerdx com Do need to specify both fusion partners in order to detect fusions No ArcherDx s Anchored Multiplex PCR AMP chemistry enables you to detect any fusion partner
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