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