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]

  1. concentrate 800ng of each samples at RT down to 3.4ul. If the samples dried up completely, resuspend in 3.4ul ddH2O per reaction
  2. mix the component according to below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
    1. 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
    2. 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
  3. prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps):
    1. add 5ul of SureSelect Oligo Capture library
    2. prepare 1:3 dilution of the RNase Block (purple cap), combine 1ul RNase Block with 3ul ddH2O
    3. add 2ul of diluted RNase Block to each capture library and mix thoroughly by pipetting
    4. keep tubes on ice
  4. prepare library for target enrichment in a separate tube (for the following steps, make sure evaporation is no more than 3-4ul):
    1. 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
    2. mix well by pipetting and cap the tubes
    3. heat up the prepped libraries + blockers at 95C 5min and 65C hold
    4. maintain the tubes at 65C while load 40ul of hybridization buffer into tube A (# of tubes needed is the # of prepped libraries)
    5. incubate both tubes at 65C for at least 5 min before go on
  5. 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)
  6. while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample
  7. while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample
  8. mix well by pipetting and the hybridization mix should be around 27-29ul
  9. add 20ul of mineral oil to each sample
  10. 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]

  1. prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect"
  2. resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen)
  3. for each hybridization, add 50ul dynalbead to 1.5ml tube
  4. wash the beads:
    1. add 200ul of SureSelect Binding Buffer
    2. mix the beads on a vortex for 5 sec
    3. put the tubes into a magnetic separator, remove and discard the supernatant
    4. repeat the above steps (4.1-4.3) for a total of 3 washes
  5. resuspend the beads in 200ul of SureSelect Binding buffer

Select Hybrid capture with SureSelect[edit]

  1. estimate the volume of hybridization mix that remained after 24-72hr incubation
  2. add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times
  3. incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed)
  4. briefly spin in a centrifuge
  5. separate the beads and buffer on a magnetic separator and remove the supernatant
  6. Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec
  7. incubate the samples for 15 min at RT
  8. wash the beads:
    1. separate the beads and buffer on magnetic separator and remove the supernatant
    2. resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec
    3. incubate the samples for 10min at 65C
    4. invert the tube to mix as the beads may be settled
    5. repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed
  9. mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads
  10. incubate the samples for 10 min at RT
  11. separate the beads and buffer on Dynal magnetic separator
  12. transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA
  13. 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]