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= BSPP DMR330k capture of HIV patient samples and N37 = * Continued from 2011_05_28: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-5-28]]<br> * Since sample A9-A12 had evaporation problem during capture, bisulfite conversion and capture were repeated<br> = Bisulfite conversion = {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID in Zhang lab''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''H20 (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount for bis-cvt (ng)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | A9||100.00||8.00||12.00||800.00||130.00||150.00 |- | A10||100.00||8.00||12.00||800.00||130.00||150.00 |- | A11||100.00||8.00||12.00||800.00||130.00||150.00 |- | A12||100.00||8.00||12.00||800.00||130.00||150.00 |}<br> * Eluted volume 10ul * After elution, the bis-cvt gDNA of each sample was combined to the previous batch and determined concentration by Qubit ss-DNA assay == Qubit ss-DNA assay == * Total amount derived from bis-cvt was not determined since each sample had different volumes <br> * Dilution factor = 200 {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc. in Qubit (ng/mL)''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''volume for 400ng''' |- | A9||340.00||68.00||5.88 |- | A10||174.00||34.80||11.49 |- | A11||138.00||27.60||14.49 |- | A12||207.00||41.40||9.66 |} <br> == Capture set up == * Note: Since capture few samples, probe S1 and S2 were diluted to 1ng/ul <br> === Probe calculation === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Subset ID''' | align="center" style="background:#f0f0f0;"|'''Adjusted Multiplier based on qPCR (Multiplier E)''' | align="center" style="background:#f0f0f0;"|'''Actual amount of probe required (probe:target ratio, 100:1)''' | align="center" style="background:#f0f0f0;"|'''Multiplier E x amount probe required''' | align="center" style="background:#f0f0f0;"|'''Probe concentration (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 5rxn (ul)''' |- | DMR.s1||0.05||2.23||0.11||1.00||0.11||0.56 |- | DMR.s2||0.07||2.23||0.16||1.00||0.16||0.81 |- | DMR.s3||0.23||18.90||4.41||23.20||0.19||0.95 |- | DMR.s4||1.00||18.90||18.90||23.20||0.81||4.07 |- | DMR.s5||4.83||18.90||91.29||29.20||3.13||15.63 |- | DMR.Exp1||0.23||18.90||4.44||31.88||0.14||0.70 |- | DMR.Exp2||0.19||18.90||3.50||24.11||0.15||0.73 |- | DMR.Exp3||0.27||18.90||5.03||28.90||0.17||0.87 |- | 105nM Oligo suppressor||||||||||1.00||5.00 |- | ||||||||Total volume||5.86||29.31 |}<br> === Reaction mix === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''5rxn mix (ul)''' |- | 10X AmpLigase Buffer||2.50||12.50 |- | DMR330k probe mix (ul)||4.86||24.30 |- | 105nM Oligo suppressor||1.00||5.00 |- | Total volume (ul)||8.36||41.80 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''sample ID''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 400ng(ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount of bis-cvt gDNA (ng)''' | align="center" style="background:#f0f0f0;"|'''Probe mix (ul)''' | align="center" style="background:#f0f0f0;"|'''10x Amligase buffer (ul)''' | align="center" style="background:#f0f0f0;"|'''105nM Oligo suppressor (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | A9||68.00||5.88||10.76||400.00||4.86||2.50||1.00||25.00 |- | A10||34.80||11.49||5.15||400.00||4.86||2.50||1.00||25.00 |- | A11||27.60||14.49||2.15||400.00||4.86||2.50||1.00||25.00 |- | A12||41.40||9.66||6.98||400.00||4.86||2.50||1.00||25.00 |}<br> '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_06_07) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_06_08) === SLN mix === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume (ul)''' |- | AmpliTaq Stoffel||10.00||U/ul||2.00||U/ul||2.40 |- | AmpLigase||5.00||U/ul||0.50||U/ul||1.20 |- | dNTP||1.00||mM||100||uM||1.20 |- | 10x AmpLigase Buffer||10.00||x||1.00||x||1.20 |- | H2O||||||||||6.00 |}<br> = PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX) = {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''8.5 rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||2.00||17.00 |- | 10uM AmpR6.3 (IndX, X 1,2,3,β¦,49)||2.00||0.00 |- | 50x SYBG||0.80||6.80 |- | 2x Phusion MM||50.00||425.00 |- | H2O||35.20||299.20 |- | Total||100.00||Aliquot 88ul, add 10ul template and 2ul AmpR6.3IndX |}<br> '''Program'''<br> 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min <br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''IndX''' |- | A9||AmpR6.3 Ind9 |- | A10||AmpR6.3 Ind10 |- | A11||AmpR6.3 Ind11 |- | A12||AmpR6.3 Ind12 |}<br> === Real-time PCR curves === [[File:2011_06_08 repeat A9-A12 Amp.png| 500px]]<br> * To purify the seq lib, 80ul of amplified amplicons were combined from two tubes (total volume of amplified amplicon = 160ul) * Purify seq lib with 0.8 volume Ampure beads, elute with 40ul EB<br> === PAGE quantification of amplified amplicon === * Loaded sample 0.5ul in each well [[File:ZhangLab_2 2011-06-08 18hr 39min_PQSeqLibgel1.jpg| 350px]] [[File:ZhangLab_2 2011-06-08 18hr 39min_PQSeqLibgel2-e.jpg| 350px]] [[File:ZhangLab_2 2011-06-08 18hr 48min_PQSeqLibgel3.jpg| 350px]] [[File:ZhangLab_2 2011-06-08 18hr 48min_PQSeqLibgel4.jpg| 350px]] [[File:ZhangLab_2 2011-06-08 19hr 35min_PQSeqLibGel5.jpg| 350px]]<br> * Pooled 100ng from each library and performed PAGE size selection in two 6% TBE gels (5-wells)<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''sample ID''' | align="center" style="background:#f0f0f0;"|'''Final conc (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 40ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 100ng (ul)''' |- | A1||15.09||603.74||6.63 |- | A2||22.97||918.86||4.35 |- | A3||18.00||719.92||5.56 |- | A4||16.51||660.47||6.06 |- | A5||24.73||989.32||4.04 |- | A6||30.86||1234.52||3.24 |- | A7||23.50||939.98||4.26 |- | A8||22.38||895.11||4.47 |- | A9||21.24||849.51||4.71 |- | A10||18.51||740.25||5.40 |- | A11||10.30||411.88||9.71 |- | A12||27.33||1093.35||3.66 |- | B1||26.96||1078.45||3.71 |- | B2||23.73||949.10||4.21 |- | B3||23.10||924.06||4.33 |- | B4||19.68||787.36||5.08 |- | B5||18.60||744.19||5.37 |- | B6||26.27||1050.91||3.81 |- | B7||19.49||779.46||5.13 |- | B8||22.61||904.41||4.42 |- | B9||14.81||592.30||6.75 |- | B10||16.74||669.69||5.97 |- | B11||17.34||693.60||5.77 |- | B12||22.11||884.39||4.52 |- | C1||25.48||1019.33||3.92 |- | C2||25.27||1010.98||3.96 |- | C3||20.78||831.06||4.81 |- | C4||24.40||976.12||4.10 |- | C5||18.86||754.28||5.30 |- | C6||15.65||625.97||6.39 |- | C7||12.22||488.99||8.18 |- | C8||23.21||928.35||4.31 |- | C9||19.49||779.79||5.13 |- | C10||15.08||603.09||6.63 |- | C11||15.90||635.98||6.29 |- | C12||14.50||579.81||6.90 |- | D1||15.37||614.81||6.51 |- | D2||14.60||583.90||6.85 |- | D3||18.49||739.71||5.41 |- | N37-1||22.48||899.01||4.45 |- | N37-2||15.68||627.19||6.38 |- | N37-3||21.10||844.00||4.74 |- | N37-4||27.40||1096.17||3.65 |- | N37-5||25.78||1031.03||3.88 |- | N37-6||23.84||953.70||4.19 |- | N37-7||14.43||577.18||6.93 |- | N37-8||18.99||759.54||5.27 |- | N37-9||14.57||582.72||6.86 |- | N37-10||14.04||561.79||7.12 |}<br> [[File:ZhangLab_2 2011-06-09 13hr 17min_PAGE ss LibFree.jpg| 350px]]<br> * Note: I accidentally loaded seqlib to one lane of DNA ladder * total volume of multiplexed libraries: 45ul [[File:ZhangLab_2 2011-06-09 18hr 02min_PQ-NP-HIV-N37SeqLib.jpg| 250px]]<br> * 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 27.38 ng/ul or 128.7nM (yields = 27.38 ng/ul x 45ul = 1,232.23ng)<br> * Concentration was also verified by Qubit ds-DNA HS, '''conc.: 19.76ng/ul or 92.8 nM''' (yields = 19.76ng/ul x 45ul = 889ng)<br> * The concentration for sequencing will be relied on Qubit quantification<br> * Sequencing: '''Sample ID: NP-HIV-N37.Ind1-49_June6, 2011'''
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