SureSeq User Guide - Oxford Gene Technology
Contents
1. Reagent 1x library pl _ X library ul 16x library ul Captured DNA and Bead Slurry 14 Nuclease free H2O 22 5 371 25 5x Herculase Il Reaction Buffer clear cap 10 165 100 mM dNTP Mix green cap 0 5 8 25 Herculase Il Fusion DNA Polymerase 1 16 5 red cap i Indexing Post Capture PCR Forward 1 16 5 Primer orange cap f PCR Primer Index 1 16 see Table 15 1 TOTAL 50 pl erie wl ul reaction Table 13 Components for PCR 1 Add 35 ul of the reaction mix to each well or tube 2 Add 1 ul of the appropriate index PCR Primer 1 16 Table 16 to each well and mix by pipetting remembering to use a different index primer for each sample to be sequenced in the same lane 3 Use a pipette to add 14 ul of each DNA sample and bead slurry to the relevant well or tube Mix by pipetting remembering to change pipette tips between samples to avoid cross contamination Perform PCR Set up PCR using the profile settings and number of cycles below in Table 14 1 Place the tubes in a thermal cycler and run the PCR program Version 2 February 2015 28 SureSeg M Solid Tumour Panel Handbook Step Temperature Time 1 98 2 min 2 98 30s 3 57 30s 4 72 1 min 5 Repeat Step 2 through Step 4 for a total of 16 times 6 72 10 min 7 4 Hold Table 14 PCR Profile Post capture PCR purification 1 2 8 9 10 1 Use only room tempera2. Prepare End Repair Master Mix e To process multiple samples prepare a master mix on ice The volume of each reagent to add to the master mix for processing 16 samples including excess is shown below as an example e For multiple samples prepare the reaction mix as shown in Table 3 Mix well on a vortex mixer Version 2 February 2015 13 SureSeg M Solid Tumour Panel Handbook Reagent 1x library pl Xlibrary pl 16x library pl DNA sample 48 Nuclease free H2O 35 2 580 8 10x End Repair Buffer 10 165 dNTP mix 1 6 26 4 T4 DNA Polymerase 1 16 5 Klenow DNA Polymerase 2 33 T4 Polynucleotide Kinase 2 2 36 3 TOTAL 100 858 52 ul sample Table 3 End Repair Mix 1 Add 52 ul of the reaction mix to each well or tube 2 Add 48 ul of each DNA sample to the relevant well or tube Mix by pipetting 10 3 times remembering to change pipette tips between samples Incubate in a thermal cycler for 30 min at 20 C Do not use a heated lid End repair purification Estimated time 40 min for 8 16 samples 1 2 8 9 Use only room temperature AMPure XP beads Mix the reagent well so that the reagent appears homogeneous and consistent in colour Add 180 ul of homogenous AMPure XP beads to each end repaired DNA sample in either 1 5 ml LoBind tubes or 0 2 ml tubes 96 well plate Mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 time
3. SureSeg amp oft SureSeq Solid Tumour Panel Handbook Catalogue Numbers 600070 96 reactions 600071 16 reactions SureSeg M Solid Tumour Panel Handbook Oxford Gene Technology Founded by Professor Ed Southern Oxford Gene Technology OGT world class genetics research solutions to leading clinical and academic research institutions CytoSure Class leading products and services offering the complete array solution for clinical genetics research Cytocell High quality fluorescence in situ hybridisation FISH probes for the detection of gene rearrangements related to inherited genetic disease and cancer SureSeq Delivering comprehensive high quality targeted sequencing products to clinical and academic researchers Genefficiency A tailored microarray and sequencing service enabling high throughput high quality genomic studies for a variety of applications For more information visit www ogt com Version 2 February 2015 2 SureSeg M Solid Tumour Panel Handbook Contents Introduction Pack contents Storage Safety Equipment and reagents required Data analysis software Procedure Sample preparation Hybridisation Addition of indexes by post capture PCR MiSeq sequencing Legal information Ordering information OOOO RA sf EEE NWMNM GG oo w Version 2 February 2015 3 SureSeg M Solid Tumour Panel Handbook Introduction The SureSeq Solid Tumour Panel has b
4. amounts in the final pool This requires both accurate determination of peak size bp provided by Agilent TapeStation High Sensitivity Kit and accurate determination of sample concentration ng ul provided by Life Technologies Qubit High Sensitivity assay Note The desired concentration nM of the Sequencing Pool for the MiSeq sequencer is 2nM Version 2 February 2015 30 SureSeg M Solid Tumour Panel Handbook Preparing the seguencing pool For each indexed DNA sample use the formulas below to determine the volume ul to use to generate the Sequencing Pool Formula 1 Molecular Weight MW MW Size in bp x 660 157 9 Formula 2 nmol ul nmol ul Sample concentration ng ul MW Formula 3 nmol ul to nM nM nmol ul 10 Formula 4 Volume of each Indexed DNA Sample Volume of each Stock Pool volume ul x Desired pool concentration 10nM Indexed DNA Sample Number of indexes in the pool x nM concentration of the sample 1 Adjusting the Stock Pool volume e f the Stock Pool volume is less than required add Low TE to bring the volume to increase the volume e f the Stock Pool volume is greater than required dry down and reconstitute to the required volume 2 Performa 1 in 5 dilution of the Stock Pool to generate the 2 nM Sequencing Pool 3 Analyse the Stock Pool 10nM and Sequencing Pool 2nM using an Agilent TapeStation High Sensitivity Kit to determine peak height Assess concen
5. 13 Remove 30 ul of the supernatant and transfer to a fresh 0 2 ml tube or 96 well plate The beads can be discarded at this time 14 Assess the quality and quantity of the DNA with an Agilent TapeStation and check that the electropherogram shows a distribution with a peak height between 250 and 275 bp 10 Setup the instrument and prepare the chip tape samples and ladder following manufacturer s instructions 15 Calculate microlitres of sample required for 500 750 ng minimum 250 ng to proceed to hybridisation If not enough amplified sample is available repeat the PCR with the remaining bead slurry Figure 4 Analysis of amplified prepped library DNA using an Agilent D1K ScreenTape assay The electropherogram shows a single peak in the size range of 200 to 275bp 10 IMPORTANT If the samples are not to be used immediately store at 20 C Version 2 February 2015 22 SureSeg M Solid Tumour Panel Handbook Hybridisation Before you begin e lt is highly recommended to test the hybridisation conditions thermal cycler and plasticware to ensure minimal evaporation occurs during the 24 hour incubation e To test add 27 ul of Hybridisation Buffer without DNA in each well that you might use and incubate at 65 C for 24 hours Check after 24 hours that the evaporation does not exceed 3 4 ul per well Hybridise the library Estimated time 25 hours for 8 16 samples Hands on time 60 min Preparation Remo
6. 40 lt 1 ng 10 gt 40 These percentage values will be higher for poor quality samples Table 9 Cycle numbers based on results from Agilent TapeStation post adapter ligation purification 2 Place the tubes in a thermal cycler and run the PCR program Note It is not recommended to perform gt 10 cycles of PCR as this will increase the percentage duplication Optional If a vacuum dryer is not available to concentrate samples to 147 ng ul for hybridisation DNA Clean amp Concentrator 5 Zymo Research Cat Nos D4003 D4004 D4013 amp D4014 columns can be used to concentrate samples In this case the PCR 1 purification steps are not necessary and can be replaced with the Zymo Research columns It will still be necessary to assess the quality and quantity of the DNA using the Agilent TapeStation as described above PCR 1 purification Estimated time 40 min for 8 16 samples 1 2 Use only room temperature AMPure XP beads Mix the reagent well so that the reagent appears homogeneous and consistent in colour Add a 1 8x volume of homogenous AMPure XP beads 90 ul of beads for a 50 ul PCR reaction volume and 180 ul of beads for a 100 ul PCR reaction volume to each PCR reaction in either 1 5 ml LoBind tubes or 0 2 ml tubes 96 well plate Mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2ml tubes plate Incubate at room temperature for 5 min Put the tube in
7. Oxford Gene Technology Operations Limited or its group companies owns all intellectual property rights in the design of the Product including the choice and configuration of the oligonucleotide sequences used in the Product The Product may only be reproduced or manufactured by Oxford Gene Technology Operations Limited or with its permission Contact information Oxford Gene Technology Begbroke Hill Woodstock Road Begbroke Oxfordshire OX5 1PF UK Oxford Gene Technology Operations Ltd Registered in England No 03845432 Begbroke Hill Woodstock Road Begbroke Oxfordshire OX5 1PF Tel 44 0 1865 856826 US 914 467 5285 Email products ogt com Technical support email support ogt com Web www ogt com Version 2 February 2015 34 SureSeg M Solid Tumour Panel Handbook Ordering information Product Contents Cat no SureSeg Solid Tumour Panel Enrichment baits sufficient for 96 600070 Assay 96 reactions samples SureSeg Solid Tumour Report SureSeg Solid Tumour Panel Enrichment baits sufficient for 16 600071 Assay 16 reactions samples SureSeg Solid Tumour Report Services Genefficiency SureSeg Solid Sequencing Service utilising the Enquire Tumour Panel Service SureSeg Solid Tumour Panel and user friendly and interactive Variant Analysis Report Genefficiency Whole Exome Whole Exome Sequencing featuring Enquire NGS Services OGT s user friendly and interactive Variant Analysi
8. contents are Oxford Gene Technology Operations Limited 2014 All rights reserved Reproduction of all or any substantial part of its contents in any form is prohibited except that individual users may print or save portions of the protocol for their own personal use This licence does not permit users to incorporate the material or any substantial part of it in any other work or publication whether in hard copy or electronic or any other form In particular but without limitation no substantial part of the handbook may be distributed or copied for any commercial purpose SureSeg Solid Tumour Panel Assay The purchaser has the non transferable right to use and consume the product for RESEARCH USE ONLY AND NOT FOR DIAGNOSTICS PROCEDURES It is not intended for use and should not be used for the diagnosis prevention monitoring treatment or alleviation of any disease or condition or for the investigation of any physiological process in any identifiable human or for any other medical purpose Trademarks Trademarks OGT SureSeq Genefficiency Labefficiency Oxford Gene Technology SureSelect Agilent Technologies Inc NanoDrop SpeedVac Thermo Fisher Scientific Hiseq MiSeq Illumina Inc Human Cot 1 DNA Quant iT Qubit Life Technologies Corp Covaris Covaris Inc Dynabeads Dynal Inc Ampure Beckman Coulter Inc Customer s obligations The Customer acknowledges that
9. min until the solution is clear 13 Remove 50 ul of the supernatant to a fresh 1 5 ml LoBind tube The beads can be discarded at this time Version 2 February 2015 12 SureSeg M Solid Tumour Panel Handbook 14 Assess the quality and quantity with Agilent 2200 TapeStation and check that the electropherogram shows a distribution with a peak height between 150 and 200 base pairs Figure 3 Set up the machine and prepare the chip tape samples and ladder following the manufacturer s instructions IMPORTANT If starting with less than 500 ng assess the quality and quantity using High Sensitivity kits IMPORTANT If the samples are not to be used immediately for the end repair step store them at 20 C A a N rl ee al Figure 3 Analysis of sheared DNA using an Agilent High Sensitivity D1K ScreenTape assay Size distribution with a peak between 150 to 200 bp 10 Note Post purification typically gt 25 of input DNA should be returned If yield is lower than 25 of input do not proceed with the processing until additional DNA from the same source can be added End repair Estimated time 45 min for 8 16 samples Hands on time 15 min Preparation e Remove the 10x End Repair Buffer and dNTP mix from storage 15 to 25 C and allow to come to room temperature e Remove the T4 DNA Polymerase Klenow DNA Polymerase and T4 Polynucleotide Kinase from storage 15 to 25 C and place on ice
10. red cap oP 8 25 SureSelect Hybridisation 5 82 5 Buffer 3 yellow cap i SureSelect Hybridisation Buffer 4 black cap BP 107 29 Total 24 5 20 needed 404 25 Table 10 Hybridisation Mix 3 Mix the components detailed in Table 11 to make the correct amount of Block Mix for the number of samples used Reagent 1x library pl X library pl 16x library pl SureSelect Indexing Block 1 25 41 25 green cap SureSelect Indexing Block 2 blue cap 2 5 41 25 SureSelect Indexing Block 3 brown cap oe 9 9 Total 5 6 92 4 Table 11 Block Mix 4 Add 5 6 ul of the Block Mix Table 12 to each well in row B of a 96 well 0 2 ml plate Plate 1 Mix by pipetting up and down at least 10 times 5 Seal the wells of row B with caps and put the 96 well 0 2 ml plate in the thermal cycler Do not heat the Hybridisation Mix or capture library yet only the prepped library with blockers 6 Run the following thermal cycler program a 95 C for 5 min b 65 C Hold Note Set the heated lid of the thermal cycler to 105 C to hold the temperature of the plate at 65 C 7 Maintain Plate 1 at 65 C while you load 20 ul of Hybridisation Mix per well into the A row of Plate 1 Load the number of wells in Row A according to the number of sample libraries prepared 8 Make sure that the plate is at 65 C for a minimum of 5 min before proceeding Version 2 February 2015 24 SureSeg M So
11. 12 Add 32 ul nuclease free water directly to the bead pellet mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2 ml Version 2 February 2015 29 SureSeg M Solid Tumour Panel Handbook tubes plate Incubate for 3 min at room temperature Centrifuge briefly to consolidate the sample and place on a magnetic stand rack for 2 3 min or until the solution is clear 13 Remove approximately 30 ul of the supernatant to a fresh 1 5 ml LoBind tube The beads can be discarded at this time 14 Analyse amplified product size using the Agilent TapeStation High Sensitivity Kit to determine the peak size for each DNA sample The electropherogram should show a peak height between 300 and 400 bp 10 Figure 5 Set up the instrument and prepare the chip samples and ladder following manufacturer s instructions 15 Assess the PCR yield using High Sensitivity dsDNA Qubit assay If the yield is lt 1 ng ul repeat the PCR with the remaining bead slurry IMPORTANT If the samples are not to be used immediately store at 20 C Figure 5 Analysis of amplified capture DNA using an Agilent High Sensitivity D1K ScreenTape assay The electropherogram shows a peak in the size range of approximately 300 400 bp 10 MiSeq sequencing The DNA samples prepared in the previous section Addition of Indexes by Post Capture PCR need to be combined such that each index tagged sample is present in equimolar
12. MPure XP beads appear homogeneous and consistent in colour Dispense 180 ul of homogenous AMPure XP beads to a 1 5 ml LoBind tube and add each 130 ul sheared DNA sample Instead add 99 ul of homogenous AMPure XP beads when starting with 55 ul of sheared sample Mix well on a vortex mixer and incubate for 5 min Put the tube in the magnetic stand and wait for the solution to clear approx 3 5 min Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 500 ul of 70 ethanol in each tube Let the tube sit for 1 min to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 step once After the second wash spin the tube briefly and return to the magnetic rack Ensure that all ethanol is completely removed using a P20 pipette and tip to remove any remaining ethanol 10 Dry the samples in a 37 C heating block thermal cycler for 3 5 min or until the residual ethanol completely evaporates IMPORTANT Do not over dry as this will decrease yield Note Bead pellet is dry when the appearance of the surface changes from shiny to matt 11 Add 52 ul nuclease free water directly to the bead pellet mix well on a vortex mixer and incubate for 2 min at room temperature 12 Spin the tube briefly and place on the magnetic stand and leave for 2 3
13. amples prepare the reaction mixes as shown in Table 8 on ice and mix well on a vortex mixer 1 Add 15 ul of each DNA sample to the relevant well or tube Version 2 February 2015 19 SureSeg M Solid Tumour Panel Handbook 2 Add 85 ul of the master mix to each well or tube and mix by pipetting 10 times remembering to change pipette tips between samples Reagent 1x library pl _ X library pl 16x library ul Ligated Library 15 Nuclease free H2O 57 969 SureSelect Primer F 2 5 42 5 SureSelect Indexing Pre Capture RT ne 2 5 ARD Herculase Il 5x Reaction Buffer 20 340 dNTP Mix included with 1 17 enzyme Herculase Il Polymerase 2 34 TOTAL 100 1445 85 ul sample Table 7 Components for 100 pl volume PCR Perform PCR Set up PCR using the profile and settings as shown in Table 8 Step Temperature C Time 1 98 2 min 2 98 30 s 3 65 30 s 4 72 1 min 5 Repeat Step 2 through Step 4 for cycle number see below 6 72 10 min 7 4 Hold Table 8 PCR Profile 1 The number of cycles from step 2 to step 4 required can be determined using to the guidelines in Table 9 Version 2 February 2015 20 SureSeg M Solid Tumour Panel Handbook DNA concentration ng pl post Average expected duplication adapter Peete j m ae C N gt 20 ng 5 lt 10 9 20 ng 6 lt 10 4 8 ng 7 10 205 2 3 ng 8 20 30 1 1 9 ng 9 30
14. compromised during the previous incubation steps Maintain the plate at 65 C while you use a multi channel pipette set at 13 ul to take 13 ul of Hybridisation Mix from the A row Plate 1 and add it to the SureSeq probe mix contained in row C of Plate 1 for each sample 12 Maintain the plate at 65 C while you use a multi channel pipette set at 13 Ul to transfer the entire contents of each prepped library mix in row B Plate 1 to the solution in row C Plate 1 Mix well by slowly pipetting up and down at least 10 times 13 Seal the wells with strip caps Make sure all wells are completely sealed Version 2 February 2015 25 SureSeg M Solid Tumour Panel Handbook 14 Incubate the hybridisation mixture for 24 hours at 65 C with a heated lid at 105 C The hybridisation mixture is now 27 to 29 ul depending on degree of evaporation during the pre incubations Hybridisation wash Estimated time 2 5 hours for 8 16 samples Hands on time 2 hours Preparation e Pre warm the required volume of SureSelect Wash Buffer 2 1 75 ml per hybridisation at 65 C for at least 1 hour before use e Take the Agilent TapeStation High Sensitivity Kit amp High Sensitivity dsDNA Qubit Kit out of the fridge at least 30 min before use to allow reagents to warm to room temperature Prepare magnetic beads 1 Vigorously resuspend the Dynal MyOne Streptavidin T1 Life Technologies magnetic beads on a vortex
15. een designed by Oxford Gene Technology OGT in collaboration with leading cancer experts to deliver accurate detection of somatic variants in 60 cancer associated genes Table 1 from fresh frozen and formalin fixed paraffin embedded tissues FFPE tissue samples The assay targets the full coding exon sequences for each gene allowing the detection of novel and known variants The SureSeg Solid Tumour Panel is compatible with Illumina HiSeq and MiSeq chemistries and is intended to provide sufficient reagents to process 96 samples through the hybridisation capture process AKT1 BRCA2 FGFR2 MAP2K1 NOTCH1 SMAD4 ALK CDH1 FGFR3 MED12 NRAS SMARCA4 APC CDKN1B FOXA1 MET PDGFRA SMARCB1 AR CDKN2A GNAS MLH1 PDGFRB SMO ARIDIA CHD1 HRAS KMT2A MLL1 PIKSCA SPOP ASXL1 CTNNB1 IDH1 KMT2D MLL2 PTEN STK11 ATM DDR2 JAK2 KMT2C MLL3 PTPN11 TP53 AXL EGFR JAK3 MTOR RB1 UTX KDM6A BRAF ERBB2 KIT NF1 RET VHL BRCA1 FGFR1 KRAS NKX3 1 ROS1 ZFHX3 Table 1 The 60 genes targeted by the SureSeq Solid Tumour Panel Assay Pack contents Component Box 1 shipped on dry ice store at 80 C USB stick Contents SureSeg Solid Tumour Panel Baits SureSeg Solid Tumour Report and SureSeg Solid Tumour Panel Handbook Version 2 February 2015 4 SureSeg M Solid Tumour Panel Handbook Storage The kit should be used before the expiry date indicated on the kit label The SureS
16. eg Solid Tumour Panel baits should be stored at 809C Safety Handling of the SureSeg Solid Tumour Panel should be carried out by trained laboratory staff in accordance with good laboratory practice using the correct protective equipment such as laboratory coats safety glasses and gloves Any chemicals used are potentially hazardous Please refer to the MSDS for specific information Intended use The SureSeg Solid Tumour Panel is a Research Use Only assay that detects variants in the exon regions of the genes listed in Table 1 as well as short distances flanking these exons Variants in non coding regions of these genes beyond the short flanking zone will not be detected This kit is designed to be used by suitably trained personnel using DNA extracted from fresh frozen tissues formalin fixed paraffin embedded tissues blood or bone marrow Intended key performance specifications e Total region size 327 5 kb e Number of genes 60 full coding regions including splice sites e FFPE compatible for samples that pass QC e Sample failure rate post QC lt 4 e Highly uniform coverage gt 97 of bases covered to at least 20 of mean in validation studies e High sensitivity detection of low frequency alleles contributing down to 1 5 at minimum 1000x coverage in dilution studies DNA input The panel has been optimised to work with as little as 100 500 ng of genomic DNA from formalin fixed paraffin embedded FFPE samples a
17. gencourt AMPure XP kit Beckman Coulter Genomics cat no A63880 A63881 A63882 e Appropriate magnetic rack for 96 well microwell plates or 1 5 ml tubes e DNA Polymerase e g Herculase Il Fusion DNA Polymerase Agilent cat no 600677 600679 e Sequencing reagents required for the MiSeq HiSeq e g Illumina cat no MS 102 2002 MS 102 2022 GD 401 3001 FC 401 3001 Data analysis software Fastg files generated with the SureSeq Solid Tumour Panel can be turned into interactive reports using OGT s powerful standalone data analysis software provided with the kit or processed using your usual analysis pipeline Please contact OGT for more details Procedure Sample preparation The following section contains instructions for sample library production specific to the Illumina sequencing platform For each sample individual library preparations hybridisations and captures are performed The samples are then tagged by PCR with an index barcode sequence Version 2 February 2015 6 SureSeq Solid Tumour Panel Handbook Sample QC integrity concentration purity Process QC fragment size 150 200 bp 10 Process QC yield ng ul Process QC fragment size 250 275 bp 10 and yield ng l Process QC fragment size 300 400 bp 10 and yield ng l i Figure 1 Workflow of sample library preparation Version 2 February 2015 7 SureSeg M Solid Tumour Panel Handbook Sam
18. hermal cycler for 3 5 min or until the residual ethanol completely evaporates IMPORTANT Do not over dry as this will decrease yield Note Bead pellet is dry when the appearance of the surface changes from shiny to maitt 12 Add 15 ul nuclease free water directly to the bead pellet mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2 ml tubes plate Incubate for 3 min at room temperature Centrifuge briefly to consolidate the sample and place on a magnetic stand rack for 2 3 min or until the solution is clear Version 2 February 2015 16 SureSeg M Solid Tumour Panel Handbook 13 Remove 14 ul of the supernatant and transfer to a fresh 0 2 ml tube 96 well plate The beads can be discarded at this time IMPORTANT Proceed immediately to the next step Adapter ligation Estimated time 30 min for 8 16 samples Hands on time 15 min Preparation e Remove the 5x T4 DNA Ligase buffer and Adapter Oligo Mix from storage 15 to 25 C and allow to come to room temperature e Remove the T4 DNA Ligase from storage 15 to 25 C and place on ice Prepare Ligation Master Mix e This step requires the preparation on ice of a 10 1 molar ratio of adapter to genomic DNA fragment using the guidance in Table 5 e Prepare the reaction mixes as shown Table 5 and mix well on a vortex mixer 1x 2 3 1x 1 1 99 1x 0 5 1x 250 1x 100 1x 50 99 Seu
19. leared solution 6 Continue to keep the tube in the magnetic stand rack whilst adding A 500 ul or e 200 ul of 70 ethanol to each tube 7 Let the tube sit for 1 min to allow any disturbed beads to settle and remove the ethanol 8 Repeat step 6 and step 7 step once 9 After the second wash seal the tube or plate and centrifuge briefly 260 x g for 30 s 10 Return the tube or plate to the magnetic stand rack and wait 1 min Remove any remaining ethanol using a gel loading tip fitted to a 20 ul pipette being careful to not touch the bead pellet 11 Dry the samples at 37 C in a heating block thermal cycler for 3 5 min or until the residual ethanol completely evaporates IMPORTANT Do not over dry as this will decrease yield Note Bead pellet is dry when the appearance of the surface changes from shiny to matt 12 Add 32 ul nuclease free water directly to the bead pellet mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2 ml tubes plate Incubate for 3 min at room temperature Centrifuge briefly to consolidate the sample and place on a magnetic stand rack for 2 3 min or until the solution is clear 13 Remove 30 ul of the supernatant and transfer to a fresh 0 2 ml tube or 96 well plate The beads can be discarded at this time 14 Take 1 ul and assess quantity using the Agilent TapeStation High Sensitivity Kit Set up the instrument and prepare the chip tape samples and ladder followi
20. leet ug ug 0 99pg9 499ng 249ng ng DNA sample 13 pl 13 pl 13 pi 13 pi 13 pl 13 pl RE 15 5 pl 15 5 pl 22 5 ul 23 5 ul 24 5 ul 25 ul 5x T4 DNA ii 10 pl 10 pl 10 pl 10 pl 10 pl 10 pl Adapter Oligo Mix 10 ul 10 ul 3 ul 2 ul 1 ul 0 5 ul T4 DNA Ligase 1 5 ul 1 5 ul 1 5 ul 1 5 pl 1 5 ul 15 ul TOTAL 50 ul 50 ul 50 ul 50 ul 50 ul 50 ul Table 5 Ligation reaction mixes 1 Add 37 ul of the reaction mix to each well or tube 2 Add 13 ul of each DNA sample to each well or tube Mix by pipetting 10 times remembering to change pipette tips between samples 3 Incubate in a thermal cycler for 15 min at 20 C Do not use a heated lid Version 2 February 2015 17 SureSeg M Solid Tumour Panel Handbook Adapter ligation purification Estimated time 40 min for 8 16 samples 1 Use only room temperature AMPure XP beads 2 Mix the reagent well so that the reagent appears homogeneous and consistent in colour 3 Add 90 ul of homogenous AMPure XP beads to each adapter ligated DNA sample in either 1 5 ml LoBind tubes or 0 2 ml tubes 96 well plate Mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2 ml tubes plate Incubate at room temperature for 5 min 4 Put the tube in the magnetic stand and wait for the solution to clear which should take approximately 3 5 min 5 Keep the tube in the magnetic stand Do not touch the beads whilst carefully removing 130 ul of the c
21. lid Tumour Panel Handbook 9 Prepare on ice In row C of a second 96 well 0 2 ml plate Plate 2 prepare the SureSeg probe mix for target enrichment Load the number of wells filled in row C according to the number of libraries prepared a For each sample add 2 ul of capture probes b Use nuclease free water to prepare a dilution of the SureSelect RNase Block purple cap as listed in Table 12 Prepare enough RNase Block mix for all samples plus some excess c Add 5 ul of diluted SureSelect RNase Block to each aliquot of SureSeg probe and mix by pipetting up and down at least 10 times Reagent 1x library pl x library pl 16x library ul SureSelect RNase Block purple 05 8 25 cap Nuclease Free water 4 5 74 25 Total 5 82 5 Table 12 RNase Block Mix 10 Add the capture library mix 7 ul from Plate 2 row C to Plate 1 row C For multiple samples use a multi channel pipette to load the SureSeq probe mix into the C row of Plate 1 Keep the plate at 65 C during this time a x b c Seal the wells with strip caps using a capping tool to make sure the fit is tight Incubate the samples at 65 C for 2 min lt d Note The following steps are to be performed as quickly as is reasonably possible whilst avoiding risk of contamination It may be helpful if two people perform these steps Note Use new strip caps as the integrity of the caps can be
22. llowing you to unlock the potential of archived samples The protocol incorporates several QC steps that determine the optimal processing workflow allowing difficult samples to be recovered whilst ensuring sufficient sequencing data for confident analysis While we recommend starting with 500 ng or more of DNA from FFPE samples to limit the level of duplication we have successfully sequenced DNA from FFPE samples where less than 100 ng of high integrity starting material was available Version 2 February 2015 5 SureSeg M Solid Tumour Panel Handbook Equipment and reagents required Required not supplied e Covaris Focused ultrasonicator or equivalent e Agilent 2200 TapeStation cat no G2965A or equivalent and relevant reagents e Thermal cycler e g BioRad MJ Research DNA Engine PTC 200 or equivalent e Qubit fluorometer Life technologies cat no Q32857 e DNA LoBind Tubes Eppendorf cat no 022431021 or equivalent e Covaris microTUBES cat no 520045 e Quant iT dsDNA HS Assay Kit or Quant iT dsDNA BR Assay Kit Life Technologies cat no Q32850 Q32853 e Molecular Biology Grade 100 Ethanol Sigma Aldrich cat no E7023 or equivalent e Molecular Biology Grade water Sigma Aldrich cat no W4502 1L or equivalent e Agilent SureSelect XT Reagent Kit 16 reactions cat no G9611A for HiSeq or G9612A for MiSeq e Dynabeads MyOne Streptavidin T1 Life Technologies cat no 656 01 656 02 or 656 03 e A
23. mixer as Dynal beads settle during storage 2 Add 50 ul Dynal magnetic beads to a 1 5 ml microfuge tube for each hybridisation performed a To wash the beads add 200 ul of SureSelect Binding Buffer mix the beads on a vortex mixer for 5 s and place the tubes into a magnetic device such as the Dynal magnetic separator Life Technologies b Remove and discard the supernatant c Repeat steps a and b for a total of 3 washes 3 Resuspend the beads in 200 ul of SureSelect Binding Buffer Hybrid capture 1 For each hybridisation record the volume of liquid that remained after 24 hour incubation 2 Keep Plate 1 at 65 C in the thermal cycler while you add the hybridisation mixture directly from the thermal cycler to the bead solution Close cap and invert the tube 3 to 5 times to mix Note Excessive evaporation for example less than 20 ul remaining after hybridisation can result in sub optimal hybridisation capture performance 3 Incubate the hybrid capture bead solution on a Nutator or equivalent for 30 min at room temperature Version 2 February 2015 26 SureSeg M Solid Tumour Panel Handbook Note Make sure the sample is properly mixing in the tube 4 Briefly spin in a centrifuge 5 Separate the beads and buffer on a Dynal magnetic separator and remove the supernatant 6 Resuspend the beads in 500 ul of SureSelect Wash Buffer 1 by mixing on a vortex mixer for 5 s Note Once the supernatant is removed it is
24. n or until the solution is clear 13 Remove 30 ul of the supernatant and transfer to a fresh 0 2 ml tube or 96 well plate The beads can be discard at this time IMPORTANT If the samples are not to be used immediately for 3 end A tailing store at 20 C 3 end A Tailing Estimated time 45 min for 8 16 samples Hands on time 15 min Preparation e Remove the 10x Klenow DNA Polymerase Buffer and dATP from storage 15 to 25 C and allow to come to room temperature e Remove the Exo Klenow DNA Polymerase from storage 15 to 25 C and place on ice Prepare A Tailing Master Mix e To process multiple samples prepare a master mix on ice The master mix for processing 16 samples including excess is shown below as an example e For multiple samples prepare the reaction mix as shown in Table 4 Mix well on a vortex mixer Reagent 1x library pl x library pl 16x library pl DNA sample 30 Nuclease free H2O 11 181 5 10x Klenow DNA Polymerase Buffer 5 82 5 dATP 1 16 5 Exo Klenow DNA Polymerase gt TOTAL 50 330 20 ul sample Table 4 Adding A bases Version 2 February 2015 15 SureSeg M Solid Tumour Panel Handbook Add 20 ul of the reaction mix to each well or tube Add 30 ul of each DNA sample to the relevant well or tube Mix by pipetting 10 times remembering to change pipette tips between samples Incubate in a thermal cycler fo
25. ng manufacturer s instructions IMPORTANT If the samples are not to be used immediately store at 4 C Version 2 February 2015 18 SureSeg M Solid Tumour Panel Handbook PCR 1 Estimated time 45 90 min for 8 16 samples Hands on time 15 min Preparation e Remove the SureSelect Primer F SureSelect Indexing Pre Capture PCR R Primer Herculase Il 5x Reaction Buffer and 100mM dNTP Mix included with enzyme from storage 15 to 25 C and allow to come to room temperature e Remove the Herculase Il Polymerase from storage 15 to 25 C and place on ice Protocol for 5 8 cycles of PCR Prepare PCR Master Mix For multiple samples prepare the reaction mixes as shown in Table 6 on ice and mix well on a vortex mixer 1 Add 15 ul of each DNA sample to the relevant well or tube 2 Add 35 ul of the master mix to each well or tube and mix by pipetting 10 times remembering to change pipette tips between samples Reagent 1x library pl _ X library ul 16x library pl Ligated Library 15 Nuclease free H2O 21 357 SureSelect Primer F 1 25 21 25 SureSelect Indexing Pre Capture PCR R primer mee 2125 Herculase Il 5x Reaction Buffer 10 170 dNTP Mix included with enzyme 0 5 8 5 Herculase Il Polymerase 1 17 TOTAL 50 595 35 ul sample Table 6 Components for 50 pl volume PCR PCR 1b for 9 or 10 cycles of PCR Prepare PCR Master Mix For multiple s
26. pecify 22 Sample 2 please specify please specify A02 please specify please specify please specify please specify 23 Sample 3 please specify please specify A03 please specify please specify please specify please specify 24 etc M SampleSheet Template Sheet DEAN A al All text in red is for user and sample specific information All text in black is required to ensure that the MiSeq will recognise the file The file needs to be saved using the MiSeq Reagent Tray ID which begins with MS then has 8 numbers followed by 300V2 when running the version 2 MiSeq chemistry e g MS2016935 300V2 The file needs to be saved as a CSV Comma delimited file After the MiSeq run the software on the supplied USB stick can be used to analyse the data on a Windows desktop machine Please refer to the manuals included on the USB stick for installation and use of the software Version 2 February 2015 32 SureSeg M Solid Tumour Panel Handbook The nucleotide seguences for the 16 indexes provided are detailed in the table below Index Number Seguence 1 ATCACG 2 CGATGT 3 TTAGGC 4 TGACCA 5 ACAGTG 6 GCCAAT 7 CAGATC 8 ACTTGA 9 GATCAG 10 TAGCTT 11 GGCTAC 12 CTTGTA 13 AAACAT 14 CAAAAG 15 GAAACC 16 AAAGCA Table 15 Index sequences Version 2 February 2015 33 SureSeg M Solid Tumour Panel Handbook Legal information This handbook and its
27. ple throughput Low throughput LT protocol All incubations are performed in 0 2 ml tubes Post incubation each reaction volume is transferred to a fresh 1 5 ml tube and sample clean up performed using the volumes highlighted in blue marked with a A All clean up steps are performed using a magnetic rack capable of holding 1 5 ml tubes High throughput HT protocol All incubations are performed in 0 2 ml tubes Post incubation the sample clean up is performed in the same 0 2 ml tubes using volumes highlighted in red marked with a e All clean up steps are performed using a magnetic rack capable of holding 0 2 ml tubes or 96 well plate 0 2 ml volume Sample QC Testing sample integrity concentration and purity DNA Integrity Use Agilent Genomic DNA ScreenTape cat no 5067 5365 and Genomic DNA Reagents cat no 5067 5366 or similar Concentration Use Invitrogen Qubit or similar Purity Use Thermo Scientific NanoDrop or similar OD 260 280 ratio of 1 8 to 2 0 and OD 260 230 ratio of 1 5 to 1 8 A requirement when working with FFPE DNA is that the fragment size peaks at a value of gt 1000 bp see below t An OD 260 280 ratio of 1 8 to 2 0 and OD 260 230 ratio of 1 5 to 1 8 is essential if omitting the post shear clean up step Use of DNA samples with lower ratios may result in poor performance If either ratio is not as recommended then use standard shearing in 130 ul followed by clean up DNA integ
28. r 30 min at 37 C If using a thermal cycler with a heated lid ensure that the lid temperature does not exceed 50 C A tailing purification Estimated time 40 min for 8 16 samples 1 2 Use only room temperature AMPure XP beads Mix the reagent well so that the reagent appears homogeneous and consistent in colour Add 90 ul of homogenous AMPure XP beads to each A tailed DNA sample in either 1 5 ml LoBind tubes or 0 2 ml tubes or 96 well plate Mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2 ml tubes plate Incubate at room temperature for 5 min Place the tube in the magnetic stand and wait for the solution to clear which should take approx 3 5 min Keep the tube in the magnetic stand Do not touch the beads whilst carefully removing 130 ul of the cleared solution Continue to keep the tube in the magnetic stand rack whilst adding A 500 ul or e 200 ul of 70 ethanol to each tube Let the tube sit for 1 min to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 step once After the second wash seal the tube or plate and centrifuge briefly 260 x g for 30 s 10 Return the tube or plate to the magnetic stand rack and wait 1 min Remove any remaining ethanol using a gel loading tip fitted to a 20 ul pipette being careful to not touch the bead pellet 11 Dry the samples at 37 C in a heating block t
29. recommended to resuspend the beads immediately in SureSelect Wash Buffer 1 so the beads do not dry out 7 Incubate the samples for 15 min at room temperature mixing every 5 min on a vortex mixer 8 Briefly spin in a centrifuge 9 Separate the beads and buffer on a Dynal magnetic separator and remove the supernatant SureSelect Wash Buffer 1 replacing it immediately with SureSelect Wash Buffer 2 below 10 Wash the beads a Resuspend the beads in 500 ul of 65 C prewarmed SureSelect Wash Buffer 2 and mix on a vortex mixer for 5 s to resuspend the beads b Incubate the samples for 10 min at 65 C in a heating block Mix every 3 min on a vortex mixer c Briefly spin in a centrifuge d Separate the beads and buffer on a Dynal magnetic separator and remove the supernatant e Once the wash buffer has been removed add fresh buffer immediately and return the tube to the heating block f Repeat steps a to d for a total of 3 washes Note Ensure all wash buffer has been removed each time 11 Add 32 ul of nuclease free water to the beads and mix on a vortex for 5 s to resuspend the beads Version 2 February 2015 27 SureSeg M Solid Tumour Panel Handbook Addition of indexes by post capture PCR PCR 2 Estimated time 1 75 hours for 8 16 samples Hands on time 15 min Prepare PCR reaction mixes For multiple samples prepare the reaction mix as shown in Table 13 on ice Mix well ona vortex mixer
30. rity Genomic DNA TapeStation Preparation Take the Agilent Genomic DNA TapeStation Reagents and tapes out of the fridge at least 30 min before use to allow reagents to warm to room temperature Open the Agilent TapeStation controller software Load Genomic DNA ScreenTape and tips into the TapeStation When analysing 1 15 samples it is recommended to use 2 x 8 tube strip tubes If analysing gt 15 samples it is recommended to use a 96 well plate Version 2 February 2015 8 SureSeg M Solid Tumour Panel Handbook Assessing DNA integrity 1 2 No o e Add 4 ul of Genomic DNA Ladder into the first tube well of the strip tube or plate Add 10 ul of Genomic DNA Sample Buffer to as many additional tubes wells as required For each sample under assessment add 1 ul of DNA sample to 10 ul of Genomic DNA Sample Buffer Seal all the tubes wells Vortex the tubes or plate for 5 s Briefly spin down to consolidate the sample to the bottom of the tubes wells Load strip of tubes or plate into the Agilent 2200 TapeStation Highlight the required samples on the controller software and fill in the sample names in the sample sheet 8 Select Start and provide a filename to save your results 9 Check that the electropherogram shows a distribution with a peak height gt 1000 bp Figure 2 Figure 2 Assessment of DNA integrity using Genomic DNA ScreenTape Concentration Qubit Preparation Set up the Qubit soft
31. s 0 2 ml tubes plate Incubate at room temperature for 5 min Place the tube in the magnetic stand Wait for the solution to clear approx 3 5 min Keep the tube in the magnetic stand Do not touch the beads whilst carefully removing 170 ul of the cleared solution Continue to keep the tube in the magnetic stand rack whilst adding A 500 ul or e 200 ul of 70 ethanol to each tube Let the tube sit for 1 min to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 step once After the second wash seal the tube or plate and centrifuge briefly 260 x g for 30 s 10 Return the tube or plate to the magnetic stand rack and wait 1 min Remove any remaining ethanol using a gel loading tip fitted to a 20 ul pipette being careful to not touch the bead pellet Version 2 February 2015 14 SureSeg M Solid Tumour Panel Handbook 11 Dry the samples at 37 C in a heating block thermal cycler for 3 5 min or until the residual ethanol completely evaporates IMPORTANT Do not over dry as this will decrease yield Note Bead pellet is dry when the appearance of the surface changes from shiny to maitt 12 Add 32 ul nuclease free water directly to the bead pellet mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2 ml tubes plate Incubate for 3 min at room temperature Centrifuge briefly to consolidate the sample and place on a magnetic stand rack for 2 3 mi
32. s Report Genefficiency RNA Seg NGS RNA Seg Sequencing featuring OGT s Enguire Services user friendly and interactive Variant Analysis Report Genefficiency Custom NGS Custom Panel Sequencing featuring Enquire Services OGT s user friendly and interactive Variant Analysis Report and expert bait design algorithm For an up to date product list and the latest product information visit www ogt com Version 2 February 2015 35 SureSeg M Solid Tumour Panel Handbook Contact us Oxford Gene Technology Begbroke Hill Woodstock Road Begbroke Oxfordshire OX5 1PF UK T 44 0 1865 856826 US 914 467 5285 E products ogt com W www ogt com Technical support E support ogt com T 44 0 1865 856826 Item number 990162 o t Version 2 February 2015 36
33. the magnetic stand and wait for the solution to clear which should take approximately 3 5 min Keep the tube in the magnetic stand Do not touch the beads whilst carefully removing 130 ul of the cleared solution Continue to keep the tube in the magnetic stand rack whilst adding A 500 ul or e 200 ul of 70 ethanol to each tube Version 2 February 2015 21 SureSeg M Solid Tumour Panel Handbook 7 Let the tube sitfor 1 min to allow any disturbed beads to settle and remove the ethanol 8 Repeat step 6 and step 7 step once 9 After the second wash seal the tube or plate and centrifuge briefly 260 x g for 30 s 10 Return the tube or plate to the magnetic stand rack and wait 1 min Remove any remaining ethanol using a gel loading tip fitted to a 20 ul pipette being careful to not touch the bead pellet Dry the samples at 37 C in a heating block thermal cycler for 3 5 min or until the residual ethanol completely evaporates IMPORTANT Do not over dry as this will decrease yield Note Bead pellet is dry when the appearance of the surface changes from shiny to matt 1 12 Add 32 ul nuclease free water directly to the bead pellet mix well by either vortexing 1 5ml tube or pipetting up and down at least 10 times 0 2 ml tubes plate Incubate for 3 min at room temperature Centrifuge briefly to consolidate the sample and place on a magnetic stand rack for 2 3 min or until the solution is clear
34. tration ng ul yield using High Sensitivity Qubit assay 4 Use formulas 1 3 above to ensure that concentration of the Stock Pool and Sequencing Pool are as expected 10nM and 2nM respectively 5 The Sequencing Pool can now be prepared for loading on to the MiSeq 6 Set up the MiSeg following manufacturer s instructions Version 2 February 2015 31 SureSeg M Solid Tumour Panel Handbook Preparing the MiSeg SampleSheet 1 The MiSeg SampleSheet can be created in Excel using the following template SampleSheet Template Microsoft Excel Bem Fk Z E Autosum gt A j By Pera Y Good Nel 4 E Norma jar a a oF it 3 Conditional Format Calculation amp xplanato Followed Hy insert Delete Forma t Sort amp Find amp ormatting as Table nd X z 2 Clear Filter Seled style el Editing AL X k Header B c D E F G H f J K Z Header 2 IEMFileVersion 4 3 Investigator Name please specify 4 Project Name please specify 5 Experiment Name please specify 6 Date please specify 7 Workflow GenerateFASTQ 8 Application FASTQ Only 9 Assay TruSeg LT 10 Description 11 Chemistry Default 12 13 Reads 14 101 15 101 17 Settings 18 19 Data 20 Sample ID Sample Name Sample Plate Sample Well 17 index ID index Sample Project Description 21 Sample 1 please specify please specify A01 please specify please specify please specify please s
35. tube Use a tapered pipette tip to slowly transfer the 130 ul or 55 ul DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube Secure the microTUBE in the tube holder and shear the DNA with the settings in Table 2 The target peak for base pair size is 150 to 200 bp Settings Value Duty Cycle 10 Intensity 5 Cycles per Burst 200 Time 6 cycles of 1 min each Set Mode Frequency sweeping Temperature 4 7 C Table 2 Covaris shear settings Place the microTUBE into an appropriately sized tube adapter and spin in a picofuge for 5 s to collect all liquid in the bottom of the micro TUBE 7 Put the Covaris microTUBE back into the loading and unloading station 8 While keeping the snap cap on insert a tapered pipette tip through the pre split septa and then slowly remove the sheared DNA Transfer the sheared DNA into a new 1 5 ml LoBind tube Version 2 February 2015 11 SureSeg M Solid Tumour Panel Handbook IMPORTANT When starting with only 55 ul sample do not purify Assess guality and quantity using the Agilent 2200 TapeStation and proceed directly to end repair When starting with lt 1000 ng use High Sensitivity kits Note Purification post shearing is the same for both LT and HT protocols Shear purification Estimated time 40 min for 8 16 samples 1 2 Use only room temperature AMPure XP beads Mix well so that the A
36. ture AMPure XP beads Mix the reagent well so that the reagent appears homogeneous and consistent in colour Add 90 ul of homogenous AMPure XP beads to each post hybridisation PCR reaction in either 1 5 ml LoBind tubes or 0 2 ml tubes 96 well plate Mix well by either vortexing 1 5 ml tube or pipetting up and down at least 10 times 0 2 ml tubes plate Incubate at room temperature for 5 min Put the tube in the magnetic stand and wait for the solution to clear which should take approximately 3 5 min Keep the tube in the magnetic stand Do not touch the beads whilst carefully removing 130 ul of the cleared solution Continue to keep the tube in the magnetic stand rack whilst adding A 500 ul or e 200 ul of 70 ethanol to each tube Let the tube sit for 1 min to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 step once After the second wash seal the tube or plate and centrifuge briefly 260 x g for 30 s Return the tube or plate to the magnetic stand rack and wait 1 min Remove any remaining ethanol using a gel loading tip fitted to a 20 ul pipette being careful to not touch the bead pellet Dry the samples at 37 C in a heating block thermal cycler for 3 5 min or until the residual ethanol completely evaporates IMPORTANT Do not over dry as this will decrease yield Note Bead pellet is dry when the appearance of the surface changes from shiny to maitt
37. ty 1 2 3 Load 1 ul of each sample onto the pedestal Click Measure Record the readings for 260 230 260 280 and the concentration ng ul DNA shearing Estimated time 6 min shearing per sample Preparation Take the AMPure XP beads Agilent 2200 TapeStation Reagents and tapes out of the fridge at least 30 min before use to allow them to warm to room temperature Make up fresh solution of 70 ethanol using molecular biology grade ethanol and molecular biology grade water Version 2 February 2015 10 SureSeg M Solid Tumour Panel Handbook Refer to the Covaris instrument user guide for set up For example for a Covaris S2 System o Fill the Covaris tank with fresh deionized water to level 12 on the fill line label o When a Covaris microTUBE is inserted ensure the water covers the visible glass part of the tube o Set the chiller temperature to 4 C o Open the Covaris control software Degassing should start automatically but if not select the Degas button Degas the instrument for least 30 min before use Shear the DNA 1 2 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample Dilute the desired amount of gDNA with 1x Low TE Buffer in a 1 5 ml LoBind tube to a total volume of 130 ul or for gDNA that has passed the purity QC criteria the DNA can be made up to 55 ul with 1x Low TE Buffer Put a Covaris microTUBE into the loading and unloading station Keep the cap on the
38. ve the SureSelect Hybridisation Buffer 3 yellow cap SureSelect Indexing Block 1 green cap SureSelect Indexing Block 2 blue cap and SureSelect Indexing Block 3 brown cap from storage 15 to 25 C and allow to come to room temperature Remove the SureSelect RNase Block and Capture Baits from storage 15 to 25 C and place on ice For each DNA sample prepared do one hybridisation capture The hybridisation reaction requires 500 750 ng of DNA with a maximum volume of 3 4 ul Note As little as 250 ng of DNA can be used for hybridisation but this may result in a higher duplication after sequencing Protocol 1 If the DNA sample concentration is below 147 ng ul use a vacuum dryer to concentrate a 500 750 ng aliquot of the sample down to 3 4 ul at lt 45 C a Put each sample into a separate well in row B of a 96 well plate Plate 1 b If the sample dries up completely resuspend in 3 4 ul of water and mix by pipetting c If processing multiple samples adjust to equivalent volumes before concentrating 2 Prepare the components detailed in Table 10 at room temperature and incubate at 65 C whilst preparing the remaining buffers to avoid precipitation Version 2 February 2015 23 SureSeg M Solid Tumour Panel Handbook Reagent 1x library pl ____X library pl 16x library pl SureSelect Hybridisation Buffer 1 orange cap ibis 062 SureSelect Hybridisation Buffer 2
39. ware for the particular assay that is being used either dsDNA Broad Range or High Sensitivity assay Take the Life Technologies Qubit Reagents out of the fridge at least 30 min before use to allow reagents to warm to room temperature Prepare the required number of 0 5 ml tubes for standards and samples use only thin wall clear 0 5 ml PCR tubes Prepare sufficient Qubit working solution for standards and samples Version 2 February 2015 9 SureSeg M Solid Tumour Panel Handbook Assessing DNA concentration 1 Dk Prepare the Qubit working solution by diluting the Qubit reagent 1 200 in Qubit buffer Load 190 ul of Qubit working solution into each of the tubes used for standards and 199 ul of Qubit working solution into each of the tubes used for samples Add 10 ul of each Qubit standard and 1 ul of sample to the appropriate tubes Mix by vortexing for 2 3 s being careful not to generate bubbles Incubate the tubes at room temperature for 2 min Measure DNA concentrations using the Qubit fluorometer following the onscreen prompts Purity NanoDrop Preparation Open the NanoDrop control software Clean the pedestals with nuclease free water Use 1 5 ul of nuclease free water to initialise the machine Blank using 1 5 ul of the relevant buffer for the samples being processed Ensure that the DNA 50 option is selected from the drop down menu on the left of the software interface Assessing DNA puri
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