High-Throughput Indexed Library Preparation And Pooled Agilent Exome Enrichment For Illumina sequencing platform September 2011 Evan Geller, gellere@mail.med.upenn.edu Schellenberg Lab, Division of Neuropathology Department of Pathology and Laboratory Medicine Perelman School of Medicine University of Pennsylvania Reagents / Consumables NEBNext Mastermix Set 1 (New England BioLabs, E6040) DNA 1000 kit (Agilent, 5067-1504) DNA High Sensitivity kit (Agilent, 5067-4626) AMPure XP Kit (Beckman Coulter, A63880) microTUBE with AFA fiber and snap cap (Covaris, 520045) 96-well 0.3mL skirtless PCR plates (E&K Scientific, 480096) 2.0mL RNase-free collection tube (Denville, C1734) 0.2mL RNase-free PCR tube (Ambion, AM12225) MicroAmp 8-cap strips (Applied Biosystems, N8010535) Elution Buffer (Qiagen, 19086) PEG 8000 (Promega, V3011) Sodium Chloride (Promega, H5271) PCR Grade water 100% ethanol Equipment Agilent 2100 Bioanalyzer Covaris Model S2 Adaptive Focused Acoustics Manual Multichannel Pipette (20uL, 200uL) 96-well magnet separator Microcentrifuge Vortex mixer Custom Oligonucleotides Order oligos HPLC purified. * corresponds to phosphorothioate bond. /5Phos/ corresponds to phosphate group. This notation is consistent with the format for IDT. P5_PCR 5’- AATGATACGGCGACCACCGAG 3’ P7_PCR 5’- CAAGCAGAAGACGGCATACGAG 3’ P5_adapter 5’- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC* T 3’ P7_adapter_# 5’- /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[Barcode]ATCTCGTATGCCGTCTTCTGCTT*G 3’ # Barcode 1 ATCACG 2 CGATGT 3 TTAGGC 4 TGACCA 5 ACAGTG 6 GCCAAT 7 CAGATC 8 ACTTGA 9 GATCAG 10 TAGCTT 11 GGCTAC 12 CTTGTA Index-Adapter Preparation Reconstitute oligonucleotides to 200 uM in 10 mM NaCl annealing buffer, then mix oligonucleotide pairs (P5_adapter, P7_adapter_#) at a 1:1 ratio (25 ul:25 ul). Heat solution to 95ºC for 5 minutes followed by cooling at a rate of 0.1ºC/s to 4ºC in a thermal cycler. Dilute with Elution Buffer to a working concentration of 15 uM. Store at -20ºC. 20% PEG / 2.5M NaCl (for SPRI purification) Add 10g PEG 8000 to a beaker containing 7.3g NaCl, bring the volume to 50mL with ddH20. Stir with a magnetic bar. Autoclave for 15 minutes, allow cooling at room temperature. Genomic Library Preparation Starting material: 260/280 ratio 1.7-1.9, 260/230 ratio >1.8 Note: Prepare all master mixes on ice. SPRI beads are retained within reaction mixture during enzyme treatments. Fragment DNA Fragmenting and size-selection are optimized for 2x100bp reads. 1ug DNA into Covaris microtube, total 50ul with Elution Buffer. Duty cycle: 10%; Intensity: 5.0; Bursts/s: 200; Duration: 120s Transfer 50ul volume into 0.3ml skirtless PCR plate; add End Repair Master Mix End Repair Master Mix Sheared DNA 50ul Elution Buffer 35ul End Repair buffer 10ul End Repair enzyme mix 5ul Total 100ul Incubate 30 min @ 20ºC Stopping point: Store at -20ºC Aliquot 150ul of AMPure XP solid-phase reversible immobilization (SPRI) beads into 0.3ml skirtless PCR plate well. Use a 200ul multichannel pipette to mix full volume 15 times; incubate at room temperature for 2 minutes. Place on 96-well plate magnet for 4 minutes. Remove supernatant and discard supernatant. With new tips and with plate on the magnet, add 100ul of 80% ethanol. Incubate 30 sec on plate magnet. Remove, discard ethanol (without disturbing beads). Place on thermal cycler for 2 minutes at 37C. Elute directly into 3’ Adenylation master mix; pipette mix 15 times to re-suspend. Seal plate with 8-well cap strips. 3’ Adenylation Master Mix Elution Buffer 42ul dA-tailing rxn buffer 5ul Klenow fragment 3ul Total 50ul Incubate 30 min @ 37ºC Note: Continue directly into clean-up and ligation of indexed-adapters. Dispense 90ul of 20% PEG, 2.5M NaCl into the sample plate. Multichannel pipette mix full volumes 15 times; incubate at room temperature for 2 minutes. Place on 96-well plate magnet for 4 minutes. Remove supernatant and discard supernatant. With new tips and with plate on the magnet, add 100ul of 80% ethanol. Incubate 30 sec on plate magnet. Remove and discard ethanol (without disturbing the beads). Place on thermal cycler for 2 minutes at 37ºC. Elute directly into Indexed-Adapter master mix; pipette mix 15 times to re-suspend. Seal plate with 8-well cap strips. Indexed-Adapter Master Mix Elution Buffer 32ul Quick ligation buffer 10ul Quick T4 DNA ligase 5ul Indexed-Adapter mix 3ul (add to each sample independently) Total 50ul Incubate 15 min @ 20ºC Stopping point: Store at -20ºC Dispense 90ul of 20% PEG, 2.5M NaCl into the sample plate. Multichannel pipette mix full volumes 15 times; incubate at room temperature for 2 minutes. Place on 96-well plate magnet for 4 minutes. Remove supernatant and discard supernatant. With new tips and with plate on the magnet, add 100ul of 80% ethanol. Incubate 30 sec on plate magnet. Remove and discard ethanol (without disturbing the beads). Place on thermal cycler for 2 minutes at 37ºC. Add 20ul of EB; pipette mix 15 times to re-suspend. Seal wells with 8-cap strip. Invitrogen E-Gel Size-Selection Load 20uL of adapter-ligated sample into each well. Do not use the outside wells in order to avoid edge effects. Load 25ul of EB to the collection wells. Run the 2% Size- selection program for a total of 21:40 minutes. Collect full volume for each sample (~10ul) and transfer to a 0.3ml skirtless PCR plate. Genomic Library Amplification Size-selected sample 10ul EB 26.5ul 5X Herculase II Buffer 10ul dNTP mix 0.5ul Herculase II Polymerase 1ul P5_PCR, 25uM (0.5uM Final) 1ul P7_PCR, 25uM (0.5uM Final) 1ul Total 50ul Profile Step 1 98ºC 2 min Step 2 98ºC 30 sec Step 3 60ºC 30 sec Step 4 72ºC 1 min Step 5 Repeat Step 2 through Step 4 for a total of 12 cycles Step 6 72ºC 5 min Step 7 4ºC forever Dispense 90ul of SPRI beads to each well of sample plate. Multichannel pipette mix full volumes 15 times; incubate at room temperature for 2 minutes. Place on 96-well plate magnet for 4 minutes. Remove supernatant and discard supernatant. With new tips and with plate on the magnet, add 100ul of 80% ethanol. Incubate 30 sec on plate magnet. Remove and discard ethanol (without disturbing the beads). Place on thermal cycler for 2 minutes at 37ºC. Add 30ul of Elution Buffer; pipette mix 15 times to re-suspend. Transfer full volume to a clean PCR plate or collection tube. Store at -20C Run samples on Bioanalyzer DNA 1000 chip to determine fragment size and concentration. Pooled Exome Enrichment In this protocol, genomic libraries representing multiple individuals are pooled together and enriched in a single exome capture. Libraries are combined evenly based on molarity from the Bioanalyzer assay. A total of 1.5 pmol prepared library per individual are placed into a single enrichment. To enrich 3 individuals in a single exome capture, 1.5 pMol of each library are pooled and lyophilized in a vacuum concentrator. A novel indexed blocking scheme (see below) is used to prevent non-specific pull down and maintain index integrity. The Indexed Blocking Reagent (IBR) replaces SureSelect Block #3 during hybridization. Indexed Blocking Oligos P5_b1_f 5’ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3’ P5_b1_r 5’ AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT 3’ P7_b1_f 5’ CAAGCAGAAGACGGCATACGAG 3’ P7_b1_r 5’ CTCGTATGCCGTCTTCTGCTTG 3’ P7_b2_f 5’ GTGACTGGAGTTCAGACGTGTGCTCTTCCGAT/3ddC/ 3’ P7_b2_r 5’ AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC 3’ Order oligos HPLC purified. /3ddC/ corresponds to 3’ dideoxyCytosine. This notation is consistent with the format for IDT. Indexed Blocking Reagent (IBR) Reconstitute oligos to 300uM with PCR Grade water. Combine equal volumes of each oligo to make 50uM indexed blocking reagent. Store at -20ºC Hybridize the Pooled Library Three components of hybridization reaction: Hybridization Buffer SureSelect Block Mix Exome Capture (plus RNAse dilution) Hybridization Buffer Reagent Volume (13ul/capture) SureSelect Hyb #1 25ul SureSelect Hyb #2 1ul SureSelect Hyb #3 10ul SureSelect Hyb #4 13ul Total 49ul Prepare in a 1.5mL RNAse-free microfuge tube. Warm to 65ºC with a heat block, placed near the hybridization thermal cycler. SureSelect Block Mix Reagent Volume per capture SureSelect Block #1 2.5ul SureSelect Block #2 2.5ul IBR 0.6ul Total 5.6ul Prepare in 0.2ml RNase-free PCR tube. Keep on ice. This will become the combined genomic library and block mix. Be mindful when sealing the cap; a crunched lid will allow volume to evaporate during hybridization. Capture Library Mix Reagent Volume per capture Exome library 5ul PCR Grade water 1ul RNase Block 1ul Total 7ul Prepare in 0.2ml RNase-free PCR tube. Keep on ice. Hybridization Reaction Reconstitute the lyophilized genomic libraries with 3.4ul of PCR Grade water. Transfer full volume to the SureSelect block mix. Mix by pipetting. Load the combined genomic library and block mix onto the thermal cycler and run the following program: Step 1 95ºC 5 min Step 2 65ºC ∞ Use a heated lid; once the combined genomic library and block mix has been at 65ºC for 10 min, place the capture library mix on the thermal cycler. After 2 additional minutes, aliquot 13ul of the hybridization buffer (at 65ºC on heat block) to the capture library mix and immediately transfer the total volume to the combined genomic library and block mix. Incubate at 65ºC for 24 hours. Wash and Amplify the Exome Enriched library Note: SureSelect Wash Buffer #2 must be warmed to 65ºC for at least 30 mins. Prepare Strept-avidin Beads For each capture; add 50ul of Dynal magnetic beads to a 2.0mL RNase-free collection tube. Add 100ul of SureSelect Binding Buffer, mix and vortex. Place on magnetic separator, remove and discard the supernatant. Repeat for a total of 3 washes. Resuspend in 100ul SureSelect Binding Buffer Wash the exome enrichment Transfer 29ul of hybridization mixture and add to strept-avidin beads. Incubate on a Nutator for 30 minutes at room temperature. Place on a magnetic separator and discard the supernatant. Add 500ul of SureSelect Wash Buffer #1, vortex and incubate for 15 minutes at room temperature. Place on a magnetic separator and discard the supernatant. Resuspend in 500ul of prewarmed Wash Buffer #2. Vortex and incubate at 65ºC for 10 minutes with a heat block (add water to the bottom of each well). Place on a magnetic separator and discard the supernatant. Repeat for a total of (3) 500ul washes. Resuspend in PCR master mix and transfer to 0.3ml skirtless PCR plate. Exome Library Amplification EB 36.5ul 5X Herculase II Buffer 10ul dNTP mix 0.5ul Herculase II Polymerase 1ul P5_PCR, 25uM (0.5uM Final) 1ul P7_PCR, 25uM (0.5uM Final) 1ul Total 50ul Profile Step 1 98ºC 2 min Step 2 98ºC 30 sec Step 3 60ºC 30 sec Step 4 72ºC 1 min Step 5 Repeat Step 2 through Step 4 for a total of 8 cycles Step 6 72ºC 5 min Step 7 4ºC forever Dispense 90ul of SPRI beads to each well of sample plate. Add full volume of PCR reaction (including strept-avidin beads) and pipette mix 15 times; incubate at room temperature for 2 minutes. Place on 96-well plate magnet for 4 minutes. Remove supernatant and discard supernatant. With new tips and with plate on the magnet, add 100ul of 80% ethanol. Incubate 30 sec on plate magnet. Remove, discard ethanol (without disturbing beads). Place on thermal cycler for 2 minutes at 37ºC. Add 30ul of Elution Buffer; pipette mix 15 times to re-suspend. Transfer full volume to a 0.3ml skirtless PCR plate. Store at -20ºC Run samples on Bioanalyzer High-Sensitivity chip to determine fragment size and concentration. Submission to sequencing facility Samples processed through a pooled-enrichment are combined with other pooled- enrichments to create a single mixture representing many individuals, each with a unique barcode. It is best to pool libraries evenly based on molarity from the Bioanalyzer High- Sensitivity assay. This mixture is diluted to 10nM and submitted for sequencing across multiple channels.