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= SeqCap Epi probe pool (UMR v1) hybridization capture on cancer patient WGBS libraries (plasma/tumor tissue DNA) = [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> === I WILL ADD ALL EXPERIMENTAL PROCEDURES SHORTLY === == <span style="color:blue">Part V: Amplifying Captured DNA Using LM-PCR</span> == ==== QUCIK RUN PCR to monitor cycle number ==== '''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 1ΰΈ and 1ΰΈΈ 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| 450px]] * 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 ==== '''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| 260px]] A = NP-SeqCap-pCancerSetA-Dec4 B = NP-SeqCap-pCancerSetB-Dec4 C = NP-SeqCap-tCancerSetC-Dec4 == <span style="color:blue">Information for sequencing and sample tracking</span> == === THERE ARE THREE POOLS OF SEQUENCING LIBRARIES === <br> === NP-SeqCap-pCancerSetA-Dec4 === * Average size: 300bp * Primer system: Illumina TruSeq * Sample NC-29 is normal control WGBS library {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Adaptor System''' | align="center" style="width:100px;background:#f0f0f0;"|'''Index #''' | align="center" style="width:100px;background:#f0f0f0;"|'''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''' |} <br> === NP-SeqCap-pCancerSetB-Dec4 === * Average size: 300bp * Primer system: Illumina TruSeq * Sample NC-7 is normal control WGBS library {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Adaptor System''' | align="center" style="width:100px;background:#f0f0f0;"|'''Index #''' | align="center" style="width:100px;background:#f0f0f0;"|'''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''' |} <br> === NP-SeqCap-tCancerSetC-Dec4 === * 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''' {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Adaptor System''' | align="center" style="width:100px;background:#f0f0f0;"|'''Index #''' | align="center" style="width:100px;background:#f0f0f0;"|'''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''' |}
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