Noi/NOTES/2014-12-4
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SeqCap Epi probe pool (UMR v1) hybridization capture on cancer patient WGBS libraries (plasma/tumor tissue DNA)[edit]
I WILL ADD ALL EXPERIMENTAL PROCEDURES SHORTLY[edit]
Part V: Amplifying Captured DNA Using LM-PCR[edit]
QUCIK RUN PCR to monitor cycle number[edit]
Components Volume (ul) 10ul rxn 8.5X 10ul rxn KAPA HiFi HotStart ReadyMix (2X) 50.00 5.00 42.50 TruS F/R (10uM) 5.00 0.50 4.25 H2O 5.00 0.50 4.25 'Total 60.00 6.00 Bead-bound captured DNA 40.00 Total PCR reaction 100.00 # Aliquot 6ul + 4ul bead-bound captured DNA (total 10ul) 2X # Ran PCR at 1ถ and 1ุ cycles on different thermocycler. LM-PCR program 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 15X, 16X --> 72C, 1min --> Hold @8C
- - Verify 2ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-12-07 17hr 18min quickPCR-15-16X-2ul.jpg
- It looked like 15 cycles gave very high amount of PCR product. I will reduce PCR cycle number to 14 cycle for amplification in large volume for sequencing, 100ul
Amplification in a large volume[edit]
Components Volume (ul) 3.2X 100ul rxn KAPA HiFi HotStart ReadyMix (2X) 50.00 160.00 TruS F/R (10uM) 5.00 16.00 H2O 5.00 16.00 Total 60.00 Bead-bound captured DNA 40.00 Total PCR reaction 100.00 # Aliquot 30ul + 20ul bead-bound captured DNA (total 50ul) 2X # Ran PCR at 14 cycles LM-PCR program 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 14X --> 72C, 1min --> Hold @8C
- - Purified amplified captured DNA with 1X AMPure beads
- - Elute with total volume 55ul of EB buffer
- - Verify purified sequencing library in 6% TBE gel by loading 2ul.
File:ZhangLab 2 2014-12-07 18hr 27min PAGE-verify-SeqLib 2ul.jpg A = NP-SeqCap-pCancerSetA-Dec4 B = NP-SeqCap-pCancerSetB-Dec4 C = NP-SeqCap-tCancerSetC-Dec4
Information for sequencing and sample tracking[edit]
THERE ARE THREE POOLS OF SEQUENCING LIBRARIES[edit]
NP-SeqCap-pCancerSetA-Dec4[edit]
- Average size: 300bp
- Primer system: Illumina TruSeq
- Sample NC-29 is normal control WGBS library
Sample | Adaptor System | Index # | Seq (RC) |
PC-P_3 | TruSeq | 2 | CGATGT |
PC-P_5 | TruSeq | 4 | TGACCA |
PC-P_6 | TruSeq | 5 | ACAGTG |
PC-P_7 | TruSeq | 6 | GCCAAT |
PC-P_8 | TruSeq | 7 | CAGATC |
6-P-4 | TruSeq | 12 | CTTGTA |
6-P-5 | TruSeq | 13 | AGTCAA |
6-P-8 | TruSeq | 14 | AGTTCC |
6-P-9 | TruSeq | 15 | ATGTCA |
6-P-10 | TruSeq | 16 | CCGTCC |
7-P-1 | TruSeq | 18 | GTCCGC |
7-P-2 | TruSeq | 19 | GTGAAA |
NC-29 | TruSeq | 23 | GAGTGG |
NP-SeqCap-pCancerSetB-Dec4[edit]
- Average size: 300bp
- Primer system: Illumina TruSeq
- Sample NC-7 is normal control WGBS library
Sample | Adaptor System | Index # | Seq (RC) |
PC-P_2 | TruSeq | 1 | ATCACG |
PC-P_4 | TruSeq | 3 | TTAGGC |
PC-P_9 | TruSeq | 8 | ACTTGA |
6-P-1 | TruSeq | 9 | GATCAG |
6-P-2 | TruSeq | 10 | TAGCTT |
6-P-3 | TruSeq | 11 | GGCTAC |
7-P-3 | TruSeq | 20 | GTGGCC |
7-P-5 | TruSeq | 21 | GTTTCG |
7-P-6 | TruSeq | 22 | CGTACG |
7-P-7 | TruSeq | 23 | GAGTGG |
7-P-8 | TruSeq | 25 | ACTGAT |
7-P-10 | TruSeq | 27 | ATTCCT |
NC-7 | TruSeq | 7 | CAGATC |
NP-SeqCap-tCancerSetC-Dec4[edit]
- Average size: 350bp
- Primer system: Zhang lab N2, one library of normal control WGBS library (NC-6) used Illumina TruSeq
- Sample NC-6 is normal control WGBS library
- THIS TABLE NEED TO BE FIXED DUE TO THE WRONG INDEX ASSIGNMENT
Sample | Adaptor System | Index # | Seq (RC) |
6T-1 | Zhang lab N2 | 20 | CCTGTC |
6T-2 | Zhang lab N2 | 21 | CAACCC |
6T-3 | Zhang lab N2 | 22 | CTCGGA |
6T-4 | Zhang lab N2 | 23 | TCGAAA |
6T-5 | Zhang lab N2 | 24 | ATTCGC |
7T-1 | Zhang lab N2 | 25 | TACTGC |
7T-2 | Zhang lab N2 | 26 | TCGTGA |
7T-4 | Zhang lab N2 | 28 | AGGTGC |
7T-5 | Zhang lab N2 | 29 | ACGAAC |
PCT-1 | Zhang lab N2 | 30 | TTAGTG |
PCT-2 | Zhang lab N2 | 31 | CACCAC |
PCT-4 | Zhang lab N2 | 32 | GCAAGG |
PCT-6 | Zhang lab N2 | 33 | CGCAAC |
PCT-7 | Zhang lab N2 | 34 | AACTGA |
NC-6 | TruSeq | 6 | GCCAAT |