Noi/NOTES/2011-6-6

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BSPP DMR330k capture of HIV patient samples and N37[edit]

  • Continued from 2011_05_28: [[1]]
  • Since sample A9-A12 had evaporation problem during capture, bisulfite conversion and capture were repeated

Bisulfite conversion[edit]

Sample ID in Zhang lab Conc. (ng/ul) Volume (ul) H20 (ul) Total amount for bis-cvt (ng) CT conversion reagent (ul) 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


  • 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[edit]

  • Total amount derived from bis-cvt was not determined since each sample had different volumes
  • Dilution factor = 200
sample ID Conc. in Qubit (ng/mL) Final conc. (ng/ul) 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


Capture set up[edit]

  • Note: Since capture few samples, probe S1 and S2 were diluted to 1ng/ul

Probe calculation[edit]

Subset ID Adjusted Multiplier based on qPCR (Multiplier E) Actual amount of probe required (probe:target ratio, 100:1) Multiplier E x amount probe required Probe concentration (ng/ul) Volume (ul) 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


Reaction mix[edit]

Components 1rxn (ul) 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


sample ID Final conc. (ng/ul) Volume for 400ng(ul) H2O (ul) Total amount of bis-cvt gDNA (ng) Probe mix (ul) 10x Amligase buffer (ul) 105nM Oligo suppressor (ul) 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


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[edit]

Components Stock conc. Unit Final conc. Unit 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


PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX)[edit]

Components 1rxn 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,…,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


Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min

Sample ID IndX
A9 AmpR6.3 Ind9
A10 AmpR6.3 Ind10
A11 AmpR6.3 Ind11
A12 AmpR6.3 Ind12


Real-time PCR curves[edit]

File:2011 06 08 repeat A9-A12 Amp.png

  • 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

PAGE quantification of amplified amplicon[edit]

  • Loaded sample 0.5ul in each well

File:ZhangLab 2 2011-06-08 18hr 39min PQSeqLibgel1.jpg File:ZhangLab 2 2011-06-08 18hr 39min PQSeqLibgel2-e.jpg File:ZhangLab 2 2011-06-08 18hr 48min PQSeqLibgel3.jpg File:ZhangLab 2 2011-06-08 18hr 48min PQSeqLibgel4.jpg File:ZhangLab 2 2011-06-08 19hr 35min PQSeqLibGel5.jpg

  • Pooled 100ng from each library and performed PAGE size selection in two 6% TBE gels (5-wells)
sample ID Final conc (ng/ul) Yields in 40ul (ng) 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


File:ZhangLab 2 2011-06-09 13hr 17min PAGE ss LibFree.jpg

  • 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

  • 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)
  • Concentration was also verified by Qubit ds-DNA HS, conc.: 19.76ng/ul or 92.8 nM (yields = 19.76ng/ul x 45ul = 889ng)
  • The concentration for sequencing will be relied on Qubit quantification
  • Sequencing: Sample ID: NP-HIV-N37.Ind1-49_June6, 2011