Noi/NOTES/2014-12-5
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WGBS library prep and BSPP capture of cfDNA from Illumina
Aims
- To generate 10 WGBS libraries from ctDNA of colorectal cancer (CRC) patients from Illumina (received from Kansas University)
- To capture WGBS libraries with MONOD v5 (LMS) probe set from Zhang's lab and CRC probe set from Illumina
Sample info
Subject# | Sample_ID | Plasma volume (ul) | Size (bp) by BA | Total Yield (ng) by BA |
KUCC BRCF 011921 | 10.A13.3 | 964 | 187 | 14 |
KUCC BRCF 005881 | 9.B7.36 | 952 | 178 | 13.2 |
KUCC BRCF 007388 | 9.C28.47 | 876 | 173 | 11.9 |
KUCC BRCF 008966 | 9.I19.36 | 882 | 185 | 4.8 |
KUCC BRCF 008282 | 9.G9.25 | 670 | 191 | 9 |
KUCC BRCF 008116 | 9.F20.56 | 924 | 197 | 5.1 |
KUCC BRCF 007855 | 9.H5.58 | 914 | 223 | 8.6 |
KUCC BRCF 008472 | 9.H3.25 | 1,000 | 219 | 4.6 |
KUCC BRCF 008527 | 9.H7.69 | 1,150 | 237 | 11.1 |
KUCC BRCF 008794 | 9.I7.3 | 770 | 249 | 9.57 |
- Each sample has volume ~22ul
- Each sample has different amount of DNA and we want to use up all DNA for WGBS library prep. To make it simple for qPCR monitoring after adaptor ligation and bisulfite conversion, I re-named the sample ID and run based on the input DNA from low to high.
Subject# | Sample_ID | Total Yield (ng)by BA | Zhang lab ID |
KUCC BRCF 008472 | 9.H3.25 | 4.6 | KU_1 |
KUCC BRCF 008966 | 9.I19.36 | 4.8 | KU_2 |
KUCC BRCF 008116 | 9.F20.56 | 5.1 | KU_3 |
KUCC BRCF 007855 | 9.H5.58 | 8.6 | KU_4 |
KUCC BRCF 008282 | 9.G9.25 | 9 | KU_5 |
KUCC BRCF 008794 | 9.I7.3 | 9.57 | KU_6 |
KUCC BRCF 008527 | 9.H7.69 | 11.1 | KU_7 |
KUCC BRCF 007388 | 9.C28.47 | 11.9 | KU_8 |
KUCC BRCF 005881 | 9.B7.36 | 13.2 | KU_9 |
KUCC BRCF 011921 | 10.A13.3 | 14 | KU_10 |
Part I: WGBS library preparation using KAPA Hyper Prep Kit
End repair & dA-tailing
- Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- KAPA Hyper Prep reaction set up (exactly in the instruction):
- - 50ul of fragmented DNA
- - 7ul of End repair & dA-tailing buffer
- - 3ul of End repair & dA-tailing enzyme mix
- - Total reaction: 60ul
End repair & dA-tailing reaction mix
Components | Volume | half rxn | 11.2x rxn |
End repair/dA-tailing buffer | 7.00 | 3.50 | 39.20 |
End repair/dA-tailing enzyme mix | 3.00 | 1.50 | 16.80 |
H2O | 6.00 | 3.00 | |
Total | 10.00 | 8.00 |
Reaction set up
Sample IDs | Zhang lab ID | Volume | End repair/dA-tailing rxn mix | H2O (ul) | Total (ul) |
9.H3.25 | KU_1 | 22.00 | 8 | 0.00 | 30.00 |
9.I19.36 | KU_2 | 22.00 | 8 | 0.00 | 30.00 |
9.F20.56 | KU_3 | 22.00 | 8 | 0.00 | 30.00 |
9.H5.58 | KU_4 | 22.00 | 8 | 0.00 | 30.00 |
9.G9.25 | KU_5 | 22.00 | 8 | 0.00 | 30.00 |
9.I7.3 | KU_6 | 22.00 | 8 | 0.00 | 30.00 |
9.H7.69 | KU_7 | 22.00 | 8 | 0.00 | 30.00 |
9.C28.47 | KU_8 | 22.00 | 8 | 0.00 | 30.00 |
9.B7.36 | KU_9 | 22.00 | 8 | 0.00 | 30.00 |
10.A13.3 | KU_10 | 22.00 | 8 | 0.00 | 30.00 |
NTC | NTC | 0.00 | 8 | 25.00 | 33.00 |
- - Add 8ul of End repair/dA-tailing rxn mix to each tube in 8-tube strip
- - Transfer 22ul of ctDNA to each rxn tube.
- - Spin down & mix well by pulse-vortexing 10X & spin down
- - 20C, 30min
- - 65C, 30min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately