Alice:Exome/2012-7-6
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Prepare human genomic DNA for Exome Capture(using Agilent's SureSelect kit)[edit]
- sheared the samples at biogem on 7/3/2012 and 7/5/2012
- 5.3.2012 batch --- mouse SCNT samples
sample ID | concentration of gDNA extracted from cell pellets (ng/ul) | ul of gDNA used for shearing (3ug each) | H2O volume (ul) | N2 Index used) |
34HHF | 218.4 | 14 | 36 | N2 index 1 |
1-9 | 350 | 9 | 41 | N2 index 2 |
B-C-5 | 346.5 | 9 | 41 | N2 index 3 |
A4 | 203 | 15 | 35 | N2 index 4 |
B-1-20 | 701 | 5 | 45 | N2 index 5 |
B-1-9 | 460.8 | 7 | 43 | N2 index 6 |
IIA11 | 242.1 | 12 | 38 | N2 index 7 |
IIA12 | 549.2 | 6 | 44 | N2 index 8 |
IB7 | 196.2 | 15 | 35 | N2 index 9 |
IIB12 | 183.9 | 16 | 34 | N2 index 10 |
IID12 | 518 | 6 | 44 | N2 index 11 |
IIF12 | 171.2 | 18 | 32 | N2 index 12 |
- NEBNext® DNA Library Prep Master Mix Set for Illumina is used (E6040S)
UPDATE: Sequencing info[edit]
120727 HL127 S5 AL-Exome-34HHF_Ind1-Jul26 Zhang Lab 50+7+50 1,983,263,000 120727 HL127 S6 AL-Exome-34HHF_Ind1-Jul26 Zhang Lab 50+7+50 2,018,412,000 120727 HL127 S7 AL-Exome-34HHF_Ind1-Jul26 Zhang Lab 50+7+50 1,992,166,000 120828 HiSeqAug28 S1 AL-exome-Aug21 Biogem 110+7+110 26,105,524,000 120828 HiSeqAug28 S2 AL-exome-Aug21 Biogem 110+7+110 26,193,028,000 120828 HiSeqAug28 S3 AL-exome-Aug21 Biogem 110+7+110 26,142,910,000
End-repair Reactions[edit]
Fragmented DNA 50 ul 10X End Repair Reaction Buffer 6 ul End Repair Enzyme Mix 5 ul Keep the tube at ~20°C for 30 minutes. Ampure bead purification (1.6x) and elute in 42.5ul ddH2O Note: for blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
A-Tailing reactions[edit]
Blunt-end DNA 43 ul 10X dA-Tailing Reaction Buffer (10X) 5 ul Klenow Fragment (3’-5’ exo-) 5 ul Incubated at 37C for 30min Ampure bead purification (1.8x) and elute in 32.5ul ddH2O
Adaptor ligation[edit]
Prepare 30uM adaptors: 100uM PE-t: 30ul 100uM PE-b: 30ul 10x stoffel buffer: 10ul H2O: 30ul 94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec.
commonly used adaptors: Blunt-end adaptors: 5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH Solexa_1_up 3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH Solexa_1_lo_nop TA adaptors (for the one adaptor protocol): 5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH PE_t_adapter 3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos t_adaptor_rc_s regular Y adaptor: PE_t_adaptor(top) ACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond PE_b_adaptor(bottom) \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-phosphorylation Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
5x Ligation buffer 10ul DNA ligase 5ul DNA adaptor (30uM) 5ul A tailed DNA 30ul Incubate at room temperature for 30 minutes purify the product with Ampure beads (1X) and elute in 42ul ddH2O
PCR amplification[edit]
Ligation products 10ul 10uM PCR_F 1ul 10uM PCR_R_index 1ul (designed by Dinh: [Link] 2X Phusion HF MasterMix 25ul H2O 13ul 4 well for each sample PCR program: 98 °C 30sec -> 7 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. purify the products with Qiaquick columns and elute in 60ul total per sample Perform another bead purification (1x) for size selection purpose and elute in 42ul ddH2O total for each sample
Sample ID | Concentration (ng/ul) | amount needed for capture (ul) |
34HHF | 103.2 | 7.8 |
1-9 | 123.1 | 6.5 |
B-C-5 | 96.4 | 8.3 |
A4 | 115 | 7.0 |
B-1-20 | 117.2 | 6.8 |
B-1-9 | 99.1 | 8.1 |
IIA-11 | 105.9 | 7.6 |
IIA-12 | 101.5 | 7.9 |
IB-7 | 92 | 8.7 |
IIB-12 | 95.4 | 8.4 |
IID-12 | 88.1 | 9.1 |
IIF-12 | 97.9 | 8.2 |
PAGE validation: File:ZhangLab 2 2012-07-15 16hr 20min-cropped.jpg File:ZhangLab 2 2012-07-15 16hr 24min.jpg
library hybridization[edit]
- concentrate 800ng of each samples at RT down to 3.4ul. If the samples dried up completely, resuspend in 3.4ul ddH2O per reaction
- mix the component according to below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
- For every reaction, add 25ul of SureSelect Hyb #1, 1ul of SureSelect Hyb #2 (red cap), 10ul of SureSelect Hyb #3 (yellow cap), 13ul of SureSelect Hyb #4, total is 49ul
- For every 3 reactions, add 75ul of SureSelect Hyb #1, 3ul of SureSelect Hyb #2 (red cap), 30ul of SureSelect Hyb #3 (yellow cap), 39ul of SureSelect Hyb #4, total is 147ul
- prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps):
- add 5ul of SureSelect Oligo Capture library
- prepare 1:3 dilution of the RNase Block (purple cap), combine 1ul RNase Block with 3ul ddH2O
- add 2ul of diluted RNase Block to each capture library and mix thoroughly by pipetting
- keep tubes on ice
- prepare library for target enrichment in a separate tube (for the following steps, make sure evaporation is no more than 3-4ul):
- put 3.4ul of prepped library(800ng) into tube B add 2.5ul of SureSelect Indexing Block #1, 2.5ul of SureSelect Block #2, and 0.6ul of SureSelect Indexing Block #3
- mix well by pipetting and cap the tubes
- heat up the prepped libraries + blockers at 95C 5min and 65C hold
- maintain the tubes at 65C while load 40ul of hybridization buffer into tube A (# of tubes needed is the # of prepped libraries)
- incubate both tubes at 65C for at least 5 min before go on
- put the SureSelect Oligo Capture Library from step 3.4 into tube C and incubate at 2 min (now the thermocycler should have three tubes per sample incubating at 65C)
- while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample
- while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample
- mix well by pipetting and the hybridization mix should be around 27-29ul
- add 20ul of mineral oil to each sample
- incubate the hybridization mixture at 65C for 24 hrs with heated lid at 105C (make sure no extensive evaporation if incubating more than 24hr)
Prepare magnetic beads[edit]
- prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect"
- resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen)
- for each hybridization, add 50ul dynalbead to 1.5ml tube
- wash the beads:
- add 200ul of SureSelect Binding Buffer
- mix the beads on a vortex for 5 sec
- put the tubes into a magnetic separator, remove and discard the supernatant
- repeat the above steps (4.1-4.3) for a total of 3 washes
- resuspend the beads in 200ul of SureSelect Binding buffer
Select Hybrid capture with SureSelect[edit]
- estimate the volume of hybridization mix that remained after 24-72hr incubation
- add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times
- incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed)
- briefly spin in a centrifuge
- separate the beads and buffer on a magnetic separator and remove the supernatant
- Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec
- incubate the samples for 15 min at RT
- wash the beads:
- separate the beads and buffer on magnetic separator and remove the supernatant
- resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec
- incubate the samples for 10min at 65C
- invert the tube to mix as the beads may be settled
- repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed
- mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads
- incubate the samples for 10 min at RT
- separate the beads and buffer on Dynal magnetic separator
- transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA
- Desalt the capture solution with AMPure Bead (1.2x) and elute in 43 ul ddH2O total (combining two capture reactions per each sample set)
post-hybridization amplification and purification[edit]
- Prepare the Post-Capture PCR Master Mix in a 1.5ml tube.
- Aliquot 20μl of captured DNA as template into one PCR tubes/wells. Mix well by pipetting up and down 5 times. 2 well for each sample set
post-capture DNA: 20ul Phusion High-Fidelity PCR Master Mix (2x): 50μl PCR grade water: 26ul Syb_FP5, 100 μM (Final Conc.: 2μM): 2ul Syb_RP7, 100 μM (Final Conc.: 2μM): 2ul Total : 100ul 98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C Qiaquick column purification and elute in 50ul ddH2O total per sample
- the PAGE gel validation results can be found on 8-14-2012 wiki page: [Here]