Noi/NOTES/2011-1-3
DMR220k capture and library construction of African's gDNA from UPENN[edit]
continued from 2010_12_16: [[1]]
Capture set up[edit]
Probe/Target Ratio | 100 |
Probes size | 220,000 |
Template amount | 200ng |
Probe calculation[edit]
- use multiplier C for probe calculation. this table for 200ng probes
Subset ID | Amount probe in Multiplier C | Conc(ng/ul) | Vol. (ul) | 58 rxn mix |
DMR.s1 | 0.11 | 2.00 | 0.06 | 3.19 |
DMR.s2 | 0.21 | 2.00 | 0.11 | 6.09 |
DMR.s3 | 4.71 | 23.20 | 0.20 | 11.78 |
DMR.s4 | 18.88 | 24.80 | 0.76 | 44.15 |
DMR.s5 | 91.22 | 26.70 | 3.42 | 198.16 |
DMR.s6 | 44.78 | 27.70 | 1.62 | 93.76 |
DMR.s7 | 44.78 | 32.00 | 1.40 | 81.16 |
105nM Oligo suppressor (ul) | 1.00 | 58.00 | ||
Total volume | 8.56 | 496.29 |
Reaction mix[edit]
Components | 1rxn (ul) | 53 rxn mix (ul) | 5 rxn mix |
10x AmpLigase buffer (ul) | 2.50 | 132.50 | 12.50 |
DMR220k probe mix (ul) | 7.56 | 400.68 | 37.80 |
105nM oligo suppressor (ul) | 1.00 | 53.00 | 5.00 |
Total (ul) | 11.06 | 586.18 | 55.30 |
Samples | Samples Concentration (ng/ul) | Volume (ul) for appox. 200ng | H2O(ul) | 10x AmpLigase buffer (ul) | DMR220k probe mix (ul) | 105nM oligo suppressor | Total volume |
A01 | 81.00 | 2.47 | 11.47 | 2.50 | 7.56 | 1.00 | 25.00 |
B01 | 68.80 | 2.91 | 11.03 | 2.50 | 7.56 | 1.00 | 25.00 |
C01 | 101.60 | 1.97 | 11.97 | 2.50 | 7.56 | 1.00 | 25.00 |
D01 | 133.00 | 1.50 | 12.44 | 2.50 | 7.56 | 1.00 | 25.00 |
E01 | 123.60 | 1.62 | 12.32 | 2.50 | 7.56 | 1.00 | 25.00 |
F01 | 104.80 | 1.91 | 12.03 | 2.50 | 7.56 | 1.00 | 25.00 |
G01 | 46.60 | 4.29 | 9.65 | 2.50 | 7.56 | 1.00 | 25.00 |
H01 | 8.38 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
A02 | 79.40 | 2.52 | 11.42 | 2.50 | 7.56 | 1.00 | 25.00 |
B02 | 124.60 | 1.61 | 12.33 | 2.50 | 7.56 | 1.00 | 25.00 |
C02 | 52.20 | 3.83 | 10.11 | 2.50 | 7.56 | 1.00 | 25.00 |
D02 | 155.20 | 1.29 | 12.65 | 2.50 | 7.56 | 1.00 | 25.00 |
E02 | 107.60 | 1.86 | 12.08 | 2.50 | 7.56 | 1.00 | 25.00 |
F02 | 109.20 | 1.83 | 12.11 | 2.50 | 7.56 | 1.00 | 25.00 |
G02 | 139.00 | 1.44 | 12.50 | 2.50 | 7.56 | 1.00 | 25.00 |
H02 | 96.20 | 2.08 | 11.86 | 2.50 | 7.56 | 1.00 | 25.00 |
A03 | 127.40 | 1.57 | 12.37 | 2.50 | 7.56 | 1.00 | 25.00 |
B03 | 99.80 | 2.00 | 11.94 | 2.50 | 7.56 | 1.00 | 25.00 |
C03 | 114.40 | 1.75 | 12.19 | 2.50 | 7.56 | 1.00 | 25.00 |
D03 | 129.20 | 1.55 | 12.39 | 2.50 | 7.56 | 1.00 | 25.00 |
E03 | 148.40 | 1.35 | 12.59 | 2.50 | 7.56 | 1.00 | 25.00 |
F03 | 84.20 | 2.38 | 11.56 | 2.50 | 7.56 | 1.00 | 25.00 |
G03 | 52.00 | 3.85 | 10.09 | 2.50 | 7.56 | 1.00 | 25.00 |
H03 | 66.00 | 3.03 | 10.91 | 2.50 | 7.56 | 1.00 | 25.00 |
A04 | 78.40 | 2.55 | 11.39 | 2.50 | 7.56 | 1.00 | 25.00 |
B04 | 19.24 | 10.40 | 3.54 | 2.50 | 7.56 | 1.00 | 25.00 |
C04 | 135.20 | 1.48 | 12.46 | 2.50 | 7.56 | 1.00 | 25.00 |
D04 | 47.40 | 4.22 | 9.72 | 2.50 | 7.56 | 1.00 | 25.00 |
E04 | 63.40 | 3.15 | 10.79 | 2.50 | 7.56 | 1.00 | 25.00 |
F04 | 88.60 | 2.26 | 11.68 | 2.50 | 7.56 | 1.00 | 25.00 |
G04 | 67.60 | 2.96 | 10.98 | 2.50 | 7.56 | 1.00 | 25.00 |
H04 | 72.40 | 2.76 | 11.18 | 2.50 | 7.56 | 1.00 | 25.00 |
A05 | 105.80 | 1.89 | 12.05 | 2.50 | 7.56 | 1.00 | 25.00 |
B05 | 86.40 | 2.31 | 11.63 | 2.50 | 7.56 | 1.00 | 25.00 |
C05 | 67.60 | 2.96 | 10.98 | 2.50 | 7.56 | 1.00 | 25.00 |
D05 | 123.40 | 1.62 | 12.32 | 2.50 | 7.56 | 1.00 | 25.00 |
E05 | 90.00 | 2.22 | 11.72 | 2.50 | 7.56 | 1.00 | 25.00 |
F05 | 111.40 | 1.80 | 12.14 | 2.50 | 7.56 | 1.00 | 25.00 |
G05 | 69.80 | 2.87 | 11.07 | 2.50 | 7.56 | 1.00 | 25.00 |
H05 | 48.60 | 4.12 | 9.82 | 2.50 | 7.56 | 1.00 | 25.00 |
A06 | 64.20 | 3.12 | 10.82 | 2.50 | 7.56 | 1.00 | 25.00 |
B06 | 0.44 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
C06 | 45.20 | 4.42 | 9.52 | 2.50 | 7.56 | 1.00 | 25.00 |
D06 | 83.80 | 2.39 | 11.55 | 2.50 | 7.56 | 1.00 | 25.00 |
E06 | 120.00 | 1.67 | 12.27 | 2.50 | 7.56 | 1.00 | 25.00 |
F06 | 83.80 | 2.39 | 11.55 | 2.50 | 7.56 | 1.00 | 25.00 |
G06 | 96.80 | 2.07 | 11.87 | 2.50 | 7.56 | 1.00 | 25.00 |
H06 | 61.80 | 3.24 | 10.70 | 2.50 | 7.56 | 1.00 | 25.00 |
I also repeated bis-cvt for some samples from JHU (D1, H5 and A6) and one from UPenn (B6) which total amount was lower than 10ng.
Sample | Sample concentration (ng/ul) | volume(ul) for approx. 200ng (ul) | H2O(ul) | 10x AmpLigase buffer (ul) | DMR220k probe mix (ul) | 105nM oligo suppressor | Total volume |
D1 (JHU) | 1.51 | 10* (15.1ng) | 3.94 | 2.50 | 7.56 | 1.00 | 25.00 |
H5 (JHU) | 30.20 | 6.62 | 7.32 | 2.50 | 7.56 | 1.00 | 25.00 |
A6 (JHU) | 0.86 | 10* (8.6ng) | 3.94 | 2.50 | 7.56 | 1.00 | 25.00 |
B6 (UPENN) | 36.90 | 5.42 | 8.52 | 2.50 | 7.56 | 1.00 | 25.00 |
Note: the bis-cvt total amount of sample D1 and A6 from JHU were still very low. I think the original conc. of gDNA was lower than noted from the lab. May need to get more samples if exp doesn't go well and need to repeat.
- aliquot approx 73ul of rxn mix to strip tubes (8 tubes)
- pipette 11.06ul of rxn mix from strip tube to 96-well plate containing BIS-CVT gDNA with multi-channel pipette
- mix by pipetting, add 20ul of mineral oil
- seal the plate with microseal film
- spin capture plate down
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h (2011_01_03) -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) (2011_01_04) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_01_05)
SLN mix preparation[edit]
Components | Stock conc. | Unit | Final conc. | Unit | 62.5 rxn mix |
AmpliTaq Stoffel | 10.00 | U/ul | 2.00 | U/ul | 25.00 |
AmpLigase | 5.00 | U/ul | 0.50 | U/ul | 12.50 |
dNTP | 1.00 | mM | 50.00 | uM | 6.25 |
10x AmpLigase Buffer | 10.00 | x | 1.00 | x | 12.50 |
H2O | 68.75 |
- aliquot 15.6ul of SLN mix into strip tubes
- add 2ul to each well with multi-channel pipette
(2011_01_05)
PCR Amplification with barcoded primers (AmpR6.3IndX)[edit]
total samples 48+1NTC+4additional samples
total volume of captured DNA 29ul
Components | 1x | 55 rxn mix |
Template (captured DNA) | 10.00 | 0.00 |
10uM AmpF6.3Sol | 2.00 | 110.00 |
10uMAmpR6.3IndX(X=1,2,3,…,48) | 2.00 | 0.00 |
50x SYBG | 0.40 | 22.00 |
2x Phusion MM | 50.00 | 2750.00 |
H2O | 35.60 | 1958.00 |
total | 100.00 | aliquot 88ul, add 2ul AmpR6.3IndX, 10ul template |
Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind |
A1 | R6.3 IndX1 | A2 | R6.3 IndX9 | A3 | R6.3 IndX17 | A4 | R6.3 IndX25 | A5 | R6.3 IndX33 | A6 | R6.3 IndX41 |
B1 | R6.3 IndX2 | B2 | R6.3 IndX10 | B3 | R6.3 IndX18 | B4 | R6.3 IndX26 | B5 | R6.3 IndX34 | B6 | R6.3 IndX42 |
C1 | R6.3 IndX3 | C2 | R6.3 IndX11 | C3 | R6.3 IndX19 | C4 | R6.3 IndX27 | C5 | R6.3 IndX35 | C6 | R6.3 IndX43 |
D1 | R6.3 IndX4 | D2 | R6.3 IndX12 | D3 | R6.3 IndX20 | D4 | R6.3 IndX28 | D5 | R6.3 IndX36 | D6 | R6.3 IndX44 |
E1 | R6.3 IndX5 | E2 | R6.3 IndX13 | E3 | R6.3 IndX21 | E4 | R6.3 IndX29 | E5 | R6.3 IndX37 | E6 | R6.3 IndX45 |
F1 | R6.3 IndX6 | F2 | R6.3 IndX14 | F3 | R6.3 IndX22 | F4 | R6.3 IndX30 | F5 | R6.3 IndX38 | F6 | R6.3 IndX46 |
G1 | R6.3 IndX7 | G2 | R6.3 IndX15 | G3 | R6.3 IndX23 | G4 | R6.3 IndX31 | G5 | R6.3 IndX39 | G6 | R6.3 IndX47 |
H1 | R6.3 IndX8 | H2 | R6.3 IndX16 | H3 | R6.3 IndX24 | H4 | R6.3 IndX32 | H5 | R6.3 IndX40 | H6 | R6.3 IndX48 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x12 -> 72C 3min
File:1stepLibConstruction 2011 01 05.PNG
- from the graph, 1 or 2 additional cycles might be added in the next amplification for the rest of captured DNA.
Amplification[edit]
- amplify the rest of captured DNA 2 more tubes/sample
(2 x 48 samples = 96 rxn, prepare 98 rxn)
Components | 1x | 98 rxn mix |
Template (captured DNA) | 9.00 | 0.00 |
10uM AmpF6.3Sol | 2.00 | 196.00 |
10uMAmpR6.3IndX(X=1,2,3,…,48) | 2.00 | 0.00 |
50x SYBG | 0.40 | 39.20 |
2x Phusion MM | 50.00 | 4900.00 |
H2O | 36.60 | 3586.80 |
total | 100.00 | aliquot 89ul, add 2ul AmpR6.3IndX, 10ul template |
Plate layout
Well No. | ///1/// | ///2/// | ///3/// | ///4/// | ///5/// | ///6/// | ///7/// | ///8/// | ///9/// | ///10/// | ///11/// | ///12/// |
A | A1 | A1 | A2 | A2 | A3 | A3 | A4 | A4 | A5 | A5 | A6 | A6 |
B | B1 | B1 | B2 | B2 | B3 | B3 | B4 | B4 | B5 | B5 | B6 | B6 |
C | C1 | C1 | C2 | C2 | C3 | C3 | C4 | C4 | C5 | C5 | C6 | C6 |
D | D1 | D1 | D2 | D2 | D3 | D3 | D4 | D4 | D5 | D5 | D6 | D6 |
E | E1 | E1 | E2 | E2 | E3 | E3 | E4 | E4 | E5 | E5 | E6 | E6 |
F | F1 | F1 | F2 | F2 | F3 | F3 | F4 | F4 | F5 | F5 | F6 | F6 |
G | G1 | G1 | G2 | G2 | G3 | G3 | G4 | G4 | G5 | G5 | G6 | G6 |
H | H1 | H1 | H2 | H2 | H3 | H3 | H4 | H4 | H5 | H5 | H6 | H6 |
Label | 1 | 1 | 2 | 2 | 3 | 3 | 4 | 4 | 5 | 5 | 6 | 6 |
Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind |
A1 | R6.3 IndX1 | A2 | R6.3 IndX9 | A3 | R6.3 IndX17 | A4 | R6.3 IndX25 | A5 | R6.3 IndX33 | A6 | R6.3 IndX41 |
B1 | R6.3 IndX2 | B2 | R6.3 IndX10 | B3 | R6.3 IndX18 | B4 | R6.3 IndX26 | B5 | R6.3 IndX34 | B6 | R6.3 IndX42 |
C1 | R6.3 IndX3 | C2 | R6.3 IndX11 | C3 | R6.3 IndX19 | C4 | R6.3 IndX27 | C5 | R6.3 IndX35 | C6 | R6.3 IndX43 |
D1 | R6.3 IndX4 | D2 | R6.3 IndX12 | D3 | R6.3 IndX20 | D4 | R6.3 IndX28 | D5 | R6.3 IndX36 | D6 | R6.3 IndX44 |
E1 | R6.3 IndX5 | E2 | R6.3 IndX13 | E3 | R6.3 IndX21 | E4 | R6.3 IndX29 | E5 | R6.3 IndX37 | E6 | R6.3 IndX45 |
F1 | R6.3 IndX6 | F2 | R6.3 IndX14 | F3 | R6.3 IndX22 | F4 | R6.3 IndX30 | F5 | R6.3 IndX38 | F6 | R6.3 IndX46 |
G1 | R6.3 IndX7 | G2 | R6.3 IndX15 | G3 | R6.3 IndX23 | G4 | R6.3 IndX31 | G5 | R6.3 IndX39 | G6 | R6.3 IndX47 |
H1 | R6.3 IndX8 | H2 | R6.3 IndX16 | H3 | R6.3 IndX24 | H4 | R6.3 IndX32 | H5 | R6.3 IndX40 | H6 | R6.3 IndX48 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
(2011_05_10)
- combine 40ul from each tube (total 120ul) and purify with Ampure beads, elute wth EB 40ul
- PAGE quantification conc. in 2% agarose gel (loaded sample 2ul)
File:ZhangLab 2 2011-01-10 23hr 05min PQ UPenSeqLibA1-H3.jpg
File:ZhangLab 2 2011-01-10 23hr 05min PQ UPenSeqLibA4-H6.jpg
Note: some samples have very low concentration, the rest of PCR product was combined and purified again.
- 25ng from each library was combined and PAGE size selection with 6% TBE 5-well PAGE gel (8 lanes)
Samples | Final conc.(ng/ul) | Yields (ng) in 40ul | Volume for 25ng (ul) |
A1 | 2.69 | 107.56 | 9.30 |
E1 | 3.67 | 146.67 | 6.82 |
B1 | 2.26 | 90.44 | 11.06 |
F1 | 1.88 | 75.38 | 13.27 |
C1 | 2.30 | 92.03 | 10.87 |
G1 | 2.19 | 87.65 | 11.41 |
D1* | 2.92 | 221.85 | 8.56 |
H1 | 1.65 | 66.00 | 15.15 |
A2* | 5.21 | 395.98 | 4.80 |
E2* | 3.04 | 231.24 | 8.22 |
B2* | 3.83 | 291.35 | 6.52 |
F2* | 3.25 | 247.26 | 7.68 |
C2 | 1.35 | 54.03 | 18.51 |
G2* | 3.11 | 236.53 | 8.03 |
D2* | 2.65 | 201.76 | 9.42 |
H2* | 3.83 | 290.95 | 6.53 |
A3 | 1.51 | 60.43 | 16.55 |
E3* | 3.85 | 292.80 | 6.49 |
B3* | 4.64 | 352.73 | 5.39 |
F3* | 3.38 | 257.24 | 7.39 |
C3* | 3.49 | 265.49 | 7.16 |
G3 | 1.40 | 55.86 | 17.90 |
D3 | 1.49 | 59.58 | 16.78 |
H3* | 5.17 | 392.54 | 4.84 |
A4 | 3.75 | 150.13 | 6.66 |
A5 | 2.33 | 93.12 | 10.74 |
B4 | 1.43 | 57.09 | 17.52 |
B5 | 5.39 | 215.58 | 4.64 |
C4 | 3.15 | 126.03 | 7.93 |
C5 | 2.69 | 107.64 | 9.29 |
D4 | 1.31 | 52.36 | 19.10 |
D5 | 2.37 | 94.73 | 10.56 |
E4 | 2.57 | 102.66 | 9.74 |
E5 | 2.05 | 82.11 | 12.18 |
F4 | 2.57 | 102.84 | 9.72 |
F5 | 3.11 | 124.52 | 8.03 |
G4 | 2.69 | 107.62 | 9.29 |
G5 | 2.53 | 101.35 | 9.87 |
H4 | 3.02 | 120.80 | 8.28 |
H5 | 2.13 | 85.21 | 11.74 |
A6 | 1.80 | 72.17 | 13.86 |
E6 | 2.29 | 91.44 | 10.94 |
B6 | 2.35 | 94.11 | 10.63 |
F6* | 3.29 | 249.84 | 7.60 |
C6 | 1.58 | 63.23 | 15.82 |
G6* | 4.79 | 363.88 | 5.22 |
D6 | 1.44 | 57.79 | 17.31 |
H6 | 1.52 | 60.83 | 16.44 |
Note: * = total volume 76ul
File:ZhangLab 2 2011-01-11 19hr 47min PAGE size selection.jpg
(2011_01_12)
- perform PAGE analysis to check the size and conc. of DNA library.
File:ZhangLab 2 2011-01-12 10hr 31min PQ SeqLib copy.jpg
- 0.6 and 0.3ul of DNA was analyzed by PAGE quantification, conc.: 7.81ng/ul or 38.2nM (yield = 7.81ng/ul * 60ul = 468.6ng
- Sequencing: Sample ID: AfricanUPenn.Ind1to48-Jan3, HL086 run
- After SE sequencing in HL086 run, the amount of each library was normalized based on number of reads and new multiplex libraries were prepared for HiSeq at NHLBI
- continued on 2011_01_20: [[2]]