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]

Back to calendar

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