Noi/NOTES/2011-1-3

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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]]