Noi/NOTES/2013-7-19: Difference between revisions

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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
== Bisulfite conversion ==
== Bisulfite conversion ==
I quantified gDNA concentration by Qubit dsDNA assay. Note that the volume of each sample they sent us was variied so I can not summarize total amount of each sample. However, based on concentration by Qubit the total amount we got of each sample less than 1ug as they claimed. Therefore I may not have enough gDNA for the second trial even I used only 500ng for bisulfite conversion. Also, there were few samples which total amount is very low so total amount for bis-cvt less than 300ng.
* I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
* I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
{| {{table}}
{| {{table}}
Line 214: Line 215:
| Amount probe required||0.78||ng
| Amount probe required||0.78||ng
|}
|}
* So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 8.89ng
* So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 3.90ng
 
==== Subset B ====
==== Subset B ====
* Conc.: 57.9ng/ul (by Qubit ssDNA assay)
* Conc.: 57.9ng/ul (by Qubit ssDNA assay)
Line 384: Line 386:
* Aliquot 9ul to 8-tube strip and add 2ul to each well with multichannel pipette.
* Aliquot 9ul to 8-tube strip and add 2ul to each well with multichannel pipette.
* Note: Actually, Clontech doesn't provide the concentration of Titanium Taq. They only said 50X concentration. I assume it has the same concentration as Stoffel fragment.
* Note: Actually, Clontech doesn't provide the concentration of Titanium Taq. They only said 50X concentration. I assume it has the same concentration as Stoffel fragment.
== PCR Amplification (TEST) ==
* Before amplify with barcode primer I test if the capture work by doing PCR in small volume (25ul) with 2ul of circularized DNA
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1x rxn'''
| align="center" style="background:#f0f0f0;"|'''30 x rxn mix'''
|-
| Captured template||2.00||0.00
|-
| 10uM AmpF6.4Sol||0.50||15.00
|-
| 10uM AmpR6.3.Ind1||0.50||15.00
|-
| 2X KAPA SYBR MM||12.50||375.00
|-
| H2O||9.50||285.00
|-
| Total||25.00||
|}
* Aliquot 23ul, add 2ul of circularized DNA template
'''Program (Eppendorf Realplex'''
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
* Loaded 3ul of PCR product in TBE gel
[[File:ZhangLab_2 2013-07-22 19hr 23min_TEST-BP-REAL.jpg| 300px]]
* It seem like I din't see very clear expected bands between 400-480bp, and the background is very high. I wonder if if added too much template, but for previous experiment I add 10ul of template in total 50ul, and got very clear bands.
* I TEST PCR again by varying template volume (0.5, 1,  2, and 4ul in total 25ul) to test if the volume of template interfere PCR.
* From the result, it seem like smaller volume of template show less background and stronger expected products. However, it didn't look as clean as I got from the first test experiment. I suspect that Exo I/III may not completely digest ds/ssDNA. I then tested enzyme activity by incubating equal amount of probes and bis-cvt gDNA I used in capture and I saw very clean gel. This suggested that enzymatic activity was good. I also run check circularized template in TBU gel (loaded 3ul) to see if there is the probe or gDNA left. I did not see either. However by PAGE verification, I may not be able to see the left over of probes and gDNA if the level was very low.
[[File:ZhangLab_2 2013-07-22 15hr 11min_confirmExoI-III-digestedprobes.jpg| 350px]] [[File:ZhangLab_2 2013-07-23 12hr 59min_TBU_circularized DNA.jpg| 110px]]
Gel1 = ExoI/III with equal amount of probe and bis-cvt gDNA (loaded difeerent amount)
Gel2 = 3ul of NTC and well B5 circularized DNA
* I decided to used 4% of template and perform PCR amplification in total 100ul reaction to make sequencing libraries.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1x rxn'''
| align="center" style="background:#f0f0f0;"|'''38 rxn mix'''
|-
| Captured template||4.00||0.00
|-
| 10uM AmpF6.4Sol||2.00||76.00
|-
| 10uM AmpR6.3Sol||2.00||0.00
|-
| 2X KAPA SYBR MM||50.00||1900.00
|-
| H2O||42.00||1596.00
|-
| Total||100.00||
|}
'''Program (Eppendorf Realplex'''
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
* Purified PCR product with 0.7 volume Ampure beads (I dilute bead 1:1 with 20% PEG/5M NaCl before adding 0.7 volume for purification).
* Eluted with 30ul H2O
* Performed PAGE quantification by loading sample 2ul
[[File:ZhangLab_2 2013-07-23 17hr 11min_qPCR_BP-REAL_A1-H1.jpg| 400px]] [[File:ZhangLab_2 2013-07-23 17hr 11min_qPCR_BP-REAL_A2-H2.jpg| 400px]]
[[File:ZhangLab_2 2013-07-23 17hr 59min_qPCR_BP-REAL_A3-H3.jpg| 400px]] [[File:ZhangLab_2 2013-07-23 17hr 59min_qPCR_BP-REAL_A4-B5.jpg| 480px]]
==== PAGE quantification ====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well IDs'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Yields in 30ul (ng)'''
| align="center" style="background:#f0f0f0;"|'''Well IDs'''
| align="center" style="background:#f0f0f0;"|'''Pool1 volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''Amount'''
| align="center" style="background:#f0f0f0;"|'''Pool2 volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''Amount (ng)'''
|-
| A1||Index 57||6.17||185.16||A1||8.10||50.00||NA||NA
|-
| B1||Index 58||6.21||186.45||B1||8.05||50.00||NA||NA
|-
| C1||Index 59||6.42||192.60||C1||7.79||50.00||NA||NA
|-
| D1||Index 60||6.20||186.10||D1||8.06||50.00||NA||NA
|-
| E1||Index 61||6.32||189.72||E1||7.91||50.00||NA||NA
|-
| F1||Index 62||6.61||198.32||F1||7.56||50.00||NA||NA
|-
| G1||Index 63||6.65||199.44||G1||7.52||50.00||NA||NA
|-
| H1||Index 64||5.93||177.79||H1||8.44||50.00||NA||NA
|-
| A2||Index 65||4.94||148.24||A2||10.12||50.00||NA||NA
|-
| B2||Index 66||5.54||166.12||B2||9.03||50.00||NA||NA
|-
| C2||Index 67||4.93||147.94||C2||10.14||50.00||NA||NA
|-
| D2||Index 68||5.56||166.68||D2||9.00||50.00||NA||NA
|-
| E2||Index 69||4.53||135.81||E2||11.04||50.00||NA||NA
|-
| F2||Index 70||5.39||161.60||F2||9.28||50.00||NA||NA
|-
| G2||Index 71||4.99||149.63||G2||10.02||50.00||NA||NA
|-
| H2||Index 72||5.61||168.28||H2||8.91||50.00||NA||NA
|-
| A3||Index 73||1.47||44.22||A3||NA||NA||12.21||18.00
|-
| B3||Index 74||2.11||63.34||B3||23.68||50.00||NA||NA
|-
| C3||Index 75||2.81||84.45||C3||17.76||50.00||NA||NA
|-
| D3||Index 76||3.68||110.48||D3||13.58||50.00||NA||NA
|-
| E3||Index 77||3.92||117.57||E3||12.76||50.00||NA||NA
|-
| F3||Index 78||3.61||108.40||F3||13.84||50.00||NA||NA
|-
| G3||Index 79||3.68||110.29||G3||13.60||50.00||NA||NA
|-
| H3||Index 80||3.67||110.14||H3||13.62||50.00||NA||NA
|-
| A4||Index 81||0.62||18.67||A4||NA||NA||28.93||18.00
|-
| B4||Index 82||5.31||159.39||B4||9.41||50.00||NA||NA
|-
| C4||Index 83||5.10||153.05||C4||9.80||50.00||NA||NA
|-
| D4||Index 84||5.88||176.34||D4||8.51||50.00||NA||NA
|-
| E4||Index 85||5.85||175.36||E4||8.55||50.00||NA||NA
|-
| F4||Index 86||5.89||176.80||F4||8.48||50.00||NA||NA
|-
| G4||Index 87||5.89||176.78||G4||8.49||50.00||NA||NA
|-
| H4||Index 88||5.46||163.76||H4||9.16||50.00||NA||NA
|-
| A5||Index 89||5.24||157.26||A5||9.54||50.00||NA||NA
|-
| B5||Index 90||4.52||135.63||B5||11.06||50.00||NA||NA
|}
* There were two samples have very low concentration, so I pool these two libraries together and purified in on lane of 5-well TBE gel
* The rest of libraries were pooled together and purified in 2 of 5-well TBE gels.
==== PAGE size-selection ====
[[File:ZhangLab_2 2013-07-23 21hr 32min_BP-SeqLib-PAGE-SS.jpg| 400px]] [[File:ZhangLab_2 2013-07-23 21hr 36min_BP-SeqLib-PAGE-SS_DD-bisPCR_e.jpg| 170px]]
* Pool 1: Library ID: '''NP-BSPP-BP.norm-Jul19'''. The libraires were resuspended in 24ul H2O
* Pool2: Library ID: '''NP-BSPP-BP.norm_low-Jul19'''. The libraries were resuspended in 12ul H2O
* Note: I also perforedm PAGE-size selection of bis PCR performed by Dinh by pooling PCR products normalized based on qt values of qPCR. After PAGE size selection, the libraries were resuspended in 12ul (Library ID: '''DD-bisPCR-BPrecommended4-Jul19''', Index 9-20, 23-44)
* Alan will do qPCR to quantify concentration of these libraries, and note that '''DD-bisPCR-BPrecommended4-Jul19''' had extramely low concentration.
==== Sequencing record ====
* Fist Miseq run: '''MiSeqJul26, 2013'''
==== 8/01/2013 ====
* From sequencing result, seem like we didn't get a good result, and we need to repeat capture.
* I summarized the total  volume/amount/concentration of gDNA samples we received from BP project.
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Volume for 500ng'''
| align="center" style="background:#f0f0f0;"|'''WellIDs'''
| align="center" style="background:#f0f0f0;"|'''Volume left (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total amount (ng)'''
| align="center" style="background:#f0f0f0;"|'''gDNA left'''
| align="center" style="background:#f0f0f0;"|'''gDNA repeat'''
| align="center" style="background:#f0f0f0;"|'''Bis-cvt left'''
| align="center" style="background:#f0f0f0;"|'''bis-cvt repeat'''
| align="center" style="background:#f0f0f0;"|'''Bad qual'''
|-
| 1||37.71||13.26||A1||5.80||19.06||718.74||218.74||y||123.00||y||
|-
| 2||39.43||12.68||B1||6.20||18.88||744.46||244.46||y||167.20||yy||
|-
| 3||50.57||9.89||C1||9.50||19.39||980.43||480.43||yy||98.30||y||
|-
| 4||34.86||14.34||D1||5.00||19.34||674.29||174.29||||143.80||yy||x
|-
| 5||56.00||8.93||E1||9.50||18.43||1032.00||532.00||yy||117.80||y||
|-
| 6||44.29||11.29||F1||8.00||19.29||854.29||354.29||y||125.60||yy||x
|-
| 7||73.71||6.78||G1||12.00||18.78||1384.57||884.57||yy||136.00||yy||
|-
| 8||29.71||16.83||H1||3.50||20.33||604.00||104.00||||138.60||yy||
|-
| 9||40.29||12.41||A2||8.00||20.41||822.29||322.29||y||117.80||y||
|-
| 10||33.43||14.96||B2||12.50||27.46||917.86||417.86||yy||162.00||yy||x
|-
| 11||44.86||11.15||C2||8.50||19.65||881.29||381.29||y||97.78||||
|-
| 12||52.86||9.46||D2||10.00||19.46||1028.57||528.57||yy||94.14||||
|-
| 13||11.34||9.00||E2||0.00||9.00||102.09||0.00||||0.00||||x
|-
| 14||36.86||9.00||F2||0.00||9.00||331.71||0.00||||0.00||||x
|-
| 15||26.74||9.00||G2||0.00||9.00||240.69||0.00||||0.00||||
|-
| 16||50.00||9.00||H2||0.00||9.00||450.00||0.00||||10.16||||x
|-
| 17||34.00||14.71||A3||22.00||36.71||1248.00||748.00||yy||0.28||||x
|-
| 18||31.14||16.06||B3||22.00||38.06||1185.14||685.14||yy||9.12||||x
|-
| 19||26.46||18.90||C3||22.00||40.90||1082.06||582.06||yy||0.00||||x
|-
| 20||20.29||20.00||D3||18.50||38.50||781.00||375.29||y||0.00||||x
|-
| 21||18.23||20.00||E3||15.50||35.50||647.11||282.54||y||0.00||||x
|-
| 22||29.43||16.99||F3||22.00||38.99||1147.43||647.43||yy||0.00||||x
|-
| 23||47.71||10.48||G3||22.00||32.48||1549.71||1049.71||yy||31.48||||
|-
| 24||32.57||15.35||H3||23.00||38.35||1249.14||749.14||yy||106.36||y||
|-
| 25||29.14||17.16||A4||21.00||38.16||1112.00||612.00||yy||125.60||yy||
|-
| 26||19.60||20.00||B4||18.00||38.00||744.80||352.80||y||149.00||yy||x
|-
| 27||24.31||20.00||C4||19.00||39.00||948.26||461.97||yy||159.40||yy||
|-
| 28||23.66||20.00||D4||19.00||39.00||922.63||449.49||yy||258.20||yy||
|-
| 29||47.43||10.54||E4||9.00||19.54||926.86||426.86||yy||120.40||y||x
|-
| 30||76.86||6.51||F4||12.50||19.01||1460.71||960.71||yy||0.00||||x
|-
| 31||40.00||12.50||G4||7.00||19.50||780.00||280.00||y||461.00||yy||
|-
| 32||32.29||15.49||H4||4.00||19.49||629.14||129.14||||578.00||yy||x
|}
* Total amount is the amount of gDNA we got
** 4 of them (13-16) have total amount < 500ng and we don't have more gDNA/bis-cvt gDNA to repeat experiment. We may need to request more if that's possible and Dr. Zhang agree to do that.
* gDNA repeat and bis-cvt repeat mean the both gDNA and bis-cvt gDNA are enough to repeat
** y = lower than required, but expected to be enough
** yy= equal or more than enough to repeat
* I am going to do bis-cvt for all samples (except 4 samples) and summarize the list of sample that need to request more if possible.

Latest revision as of 01:27, 20 August 2013

DNA methylation assay for Blueprint project[edit]

Bisulfite conversion[edit]

I quantified gDNA concentration by Qubit dsDNA assay. Note that the volume of each sample they sent us was variied so I can not summarize total amount of each sample. However, based on concentration by Qubit the total amount we got of each sample less than 1ug as they claimed. Therefore I may not have enough gDNA for the second trial even I used only 500ng for bisulfite conversion. Also, there were few samples which total amount is very low so total amount for bis-cvt less than 300ng.

  • I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
Sample IDs WellIDs Conc. (ng/ul) Volume (ul) H2O (ul) CT conversion reagent (ul) Total volume (ul) Total amount (ng)
1 A1 37.71 13.26 6.74 130.00 150.00 500.00
2 B1 39.43 12.68 7.32 130.00 150.00 500.00
3 C1 50.57 9.89 10.11 130.00 150.00 500.00
4 D1 34.86 14.34 5.66 130.00 150.00 500.00
5 E1 56.00 8.93 11.07 130.00 150.00 500.00
6 F1 44.29 11.29 8.71 130.00 150.00 500.00
7 G1 73.71 6.78 13.22 130.00 150.00 500.00
8 H1 29.71 16.83 3.17 130.00 150.00 500.00
9 A2 40.29 12.41 7.59 130.00 150.00 500.00
10 B2 33.43 14.96 5.04 130.00 150.00 500.00
11 C2 44.86 11.15 8.85 130.00 150.00 500.00
12 D2 52.86 9.46 10.54 130.00 150.00 500.00
**13 E2 11.34 9.00 11.00 130.00 150.00 102.09
*14 F2 36.86 9.00 11.00 130.00 150.00 331.71
**15 G2 26.74 9.00 11.00 130.00 150.00 240.69
*16 H2 50.00 9.00 11.00 130.00 150.00 450.00
17 A3 34.00 14.71 5.29 130.00 150.00 500.00
18 B3 31.14 16.06 3.94 130.00 150.00 500.00
19 C3 26.46 18.90 1.10 130.00 150.00 500.00
*20 D3 20.29 20.00 0.00 130.00 150.00 405.71
*21 E3 18.23 20.00 0.00 130.00 150.00 364.57
22 F3 29.43 16.99 3.01 130.00 150.00 500.00
23 G3 47.71 10.48 9.52 130.00 150.00 500.00
24 H3 32.57 15.35 4.65 130.00 150.00 500.00
25 A4 29.14 17.16 2.84 130.00 150.00 500.00
*26 B4 19.60 20.00 0.00 130.00 150.00 392.00
27 C4 24.31 20.00 0.00 130.00 150.00 486.29
28 D4 23.66 20.00 0.00 130.00 150.00 473.14
29 E4 47.43 10.54 9.46 130.00 150.00 500.00
30 F4 76.86 6.51 13.49 130.00 150.00 500.00
31 G4 40.00 12.50 7.50 130.00 150.00 500.00
32 H4 32.29 15.49 4.51 130.00 150.00 500.00
33 A5 (12878) 58.00 8.62 11.38 130.00 150.00 500.00
34 B5 (12878) 59.00 8.47 11.53 131.00 151.00 500.00
 Program 
1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
  • Eluted bisulfite converted DNA form the bead with 20ul H2O
  • I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul.

ssDNA Qubit assay result[edit]

Sample IDs WellIDs Start amount (ng) Conc. (ng/ul) Yield in 17ul (ng) Yield left after
Qubit (15ul)
% Recovery in 17ul
1 A1 500.00 21.00 357.00 315.00 71.40
2 B1 500.00 24.40 414.80 366.00 82.96
3 C1 500.00 19.10 324.70 286.50 64.94
4 D1 500.00 22.60 384.20 339.00 76.84
5 E1 500.00 20.60 350.20 309.00 70.04
6 F1 500.00 21.20 360.40 318.00 72.08
7 G1 500.00 22.00 374.00 330.00 74.80
8 H1 500.00 22.20 377.40 333.00 75.48
9 A2 500.00 20.60 350.20 309.00 70.04
10 B2 500.00 24.00 408.00 360.00 81.60
11 C2 500.00 19.06 324.02 285.90 64.80
12 D2 500.00 18.78 319.26 281.70 63.85
13 E2 102.09 2.06 35.02 30.90 34.30
14 F2 331.71 8.08 137.36 121.20 41.41
15 G2 240.69 6.50 110.50 97.50 45.91
16 H2 450.00 12.32 209.44 184.80 46.54
17 A3 500.00 11.56 196.52 173.40 39.30
18 B3 500.00 12.24 208.08 183.60 41.62
19 C3 500.00 11.44 194.48 171.60 38.90
20 D3 405.71 7.92 134.64 118.80 33.19
21 E3 364.57 8.10 137.70 121.50 37.77
22 F3 500.00 11.38 193.46 170.70 38.69
23 G3 500.00 13.96 237.32 209.40 47.46
24 H3 500.00 19.72 335.24 295.80 67.05
25 A4 500.00 21.20 360.40 318.00 72.08
26 B4 392.00 23.00 391.00 345.00 99.74
27 C4 486.29 23.80 404.60 357.00 83.20
28 D4 473.14 31.40 533.80 471.00 112.82
29 E4 500.00 20.80 353.60 312.00 70.72
30 F4 500.00 10.46 177.82 156.90 35.56
31 G4 500.00 47.00 799.00 705.00 159.80
32 H4 500.00 56.00 952.00 840.00 190.40
33 A5 (12878) 500.00 24.00 408.00 360.00 81.60
34 B5 (12878) 500.00 24.60 418.20 369.00 83.64
  • There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting.
  • Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.

BSPP capture set up[edit]

Original set[edit]

  • Conc.: 25.4 ng/ul (by Qubit ssDNA assay)
Probe:target 500:1 '
Probe size 12000
Template 150 ng
Human gDNA MW 1.9500E+12 g/mol
Human gDNA (150ng) 7.6923E-20 mol (150ng/1.9500E+12 g/mol)
Probe required (500:1) 3.8462E-17 mol (7.6923E-20 mol * 500)
Probe MW (12,000 probes, 110nt) 4.2900E+08 g/mol (12000 probes * 110nt * 325Da/nt)
Amount probe required 1.6500E-08 g (4.2900E+08 g/mol * 3.8462E-17 mol)
Amount probe required 16.50 ng

Subset A[edit]

  • Conc.: 17.6ng/ul (by Qubit ssDNA assay)
Probe:target 500:1 '
Probe size 566 probes
Template 150 ng
Human gDNA MW 1.9500E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (200ng) 7.6923E-20 mol (200ng/1.9500E+12 g/mol)
Probe required (500:1) 3.8462E-17 mol (1.5385E-19mol * 500)
Probe MW (566 probes, 1100nt) 2.0235E+07 g/mol (566 probes * 110nt * 325Da/nt)
Amount probe required 7.7825E-10 g 2.0235E+07 g/mol * 3.8462E-17mol)
Amount probe required 0.78 ng
  • So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 3.90ng

Subset B[edit]

  • Conc.: 57.9ng/ul (by Qubit ssDNA assay)
Probe:target 500:1 '
Probe size 11397 probes
Template 150 ng
Human gDNA MW 1.9500E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (200ng) 7.6923E-20 mol (150ng/1.9500E+12 g/mol)
Probe required (500:1) 3.8462E-17 mol (1.5385E-19mol * 500)
Probe MW (11,397 probes, 1100nt) 4.0744E+08 g/mol (11397 probes * 110nt * 325Da/nt)
Amount probe required 1.5671E-08 g (4.0744E+08 g/mol * 3.8462E-17 mol)
Amount probe required 15.67 ng
  • So for 1:5:5 (Original:A:B), amount probe required = 15.67ng * 5 = 78.35ng

Summary of probes required for cpature[edit]

Probe set Conc. (ng/ul) Vmount required Volume (ul)/samples Volume for ~38x rxn
Original 25.40 16.50 0.65 24.69
Subset A 17.60 3.89 0.22 8.40
Subset B 57.90 78.35 1.35 51.42
  • I will do two captures for positive control (12878) to see the consistency between the two technical replicates and one NTC
  • I will save at least 2ul of bis-cvt DNA for bisulfite PCR if we need to do that. However, Dr. Zhang suggested us to ignore the recommended_4 site.
Sample IDs WellIDs Conc. (ng/ul) Volume required (ul) BP-O (ul) BP.norm-A (ul) BP.norm-B (ul) 10X Ampligase
Buffer (ul)
H2O (ul) Total vulome (ul) Total biscvt DNA (ng)
1 A1 21.00 7.14 0.65 0.22 1.35 2.00 8.64 20.00 150.00
2 B1 24.40 6.15 0.65 0.22 1.35 2.00 9.63 20.00 150.00
3 C1 19.10 7.85 0.65 0.22 1.35 2.00 7.93 20.00 150.00
4 D1 22.60 6.64 0.65 0.22 1.35 2.00 9.14 20.00 150.00
5 E1 20.60 7.28 0.65 0.22 1.35 2.00 8.50 20.00 150.00
6 F1 21.20 7.08 0.65 0.22 1.35 2.00 8.70 20.00 150.00
7 G1 22.00 6.82 0.65 0.22 1.35 2.00 8.96 20.00 150.00
8 H1 22.20 6.76 0.65 0.22 1.35 2.00 9.02 20.00 150.00
9 A2 20.60 7.28 0.65 0.22 1.35 2.00 8.50 20.00 150.00
10 B2 24.00 6.25 0.65 0.22 1.35 2.00 9.53 20.00 150.00
11 C2 19.06 7.87 0.65 0.22 1.35 2.00 7.91 20.00 150.00
12 D2 18.78 7.99 0.65 0.22 1.35 2.00 7.79 20.00 150.00
13 E2 2.06 13.00 0.65 0.22 1.35 2.00 2.78 20.00 26.78
14 F2 8.08 13.00 0.65 0.22 1.35 2.00 2.78 20.00 105.04
15 G2 6.50 13.00 0.65 0.22 1.35 2.00 2.78 20.00 84.50
16 H2 12.32 12.18 0.65 0.22 1.35 2.00 3.60 20.00 150.00
17 A3 11.56 12.98 0.65 0.22 1.35 2.00 2.80 20.00 150.00
18 B3 12.24 12.25 0.65 0.22 1.35 2.00 3.53 20.00 150.00
19 C3 11.44 13.00 0.65 0.22 1.35 2.00 2.78 20.00 148.72
20 D3 7.92 13.00 0.65 0.22 1.35 2.00 2.78 20.00 102.96
21 E3 8.10 13.00 0.65 0.22 1.35 2.00 2.78 20.00 105.30
22 F3 11.38 13.00 0.65 0.22 1.35 2.00 2.78 20.00 147.94
23 G3 13.96 10.74 0.65 0.22 1.35 2.00 5.04 20.00 150.00
24 H3 19.72 7.61 0.65 0.22 1.35 2.00 8.17 20.00 150.00
25 A4 21.20 7.08 0.65 0.22 1.35 2.00 8.70 20.00 150.00
26 B4 23.00 6.52 0.65 0.22 1.35 2.00 9.26 20.00 150.00
27 C4 23.80 6.30 0.65 0.22 1.35 2.00 9.48 20.00 150.00
28 D4 31.40 4.78 0.65 0.22 1.35 2.00 11.00 20.00 150.00
29 E4 20.80 7.21 0.65 0.22 1.35 2.00 8.57 20.00 150.00
30 F4 10.46 13.00 0.65 0.22 1.35 2.00 2.78 20.00 135.98
31 G4 47.00 3.19 0.65 0.22 1.35 2.00 12.59 20.00 150.00
32 H4 56.00 2.68 0.65 0.22 1.35 2.00 13.10 20.00 150.00
33 A5 (12878) 24.00 6.25 0.65 0.22 1.35 2.00 9.53 20.00 150.00
34 B5 (12878) 24.60 6.10 0.65 0.22 1.35 2.00 9.68 20.00 150.00
35 C5 (NTC) 0.00 0.00 0.65 0.22 1.35 2.00 15.78 20.00 0.00

Reaction mix[edit]

Components 1x rxn 38 rxn mix
10X Ampligase buffer 2.00 76.00
BP-O 0.65 24.70
BP.norm-A 0.22 8.36
BP.norm-B 1.35 51.30
Total 4.22 160.36
  • Aliquot ~18ul to 8-tube strip and add 4.22ul of reaction mix to the 96-well plate with bis-cvt DNA and H2O with multichannel pipette
  • Mix 15 time by pipetting
  • Add 40ul of mineral oil & seal with silicone sealing mat
  • Spin the plate and place on thermocycler
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2ul SLN mix (10x Titanium Taq; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix solution[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul), total 80ul
Titanium Taq (Klentaq fragment) 10 U/ul 2 U/ul 16.00
AmpLigase 5 U/ul 0.5 U/ul 8.00
dNTP 1 mM 100 uM 8.00
10x AmpLigase Buffer 10 x 1 x 8.00
H2O 40.00
  • Aliquot 9ul to 8-tube strip and add 2ul to each well with multichannel pipette.
  • Note: Actually, Clontech doesn't provide the concentration of Titanium Taq. They only said 50X concentration. I assume it has the same concentration as Stoffel fragment.

PCR Amplification (TEST)[edit]

  • Before amplify with barcode primer I test if the capture work by doing PCR in small volume (25ul) with 2ul of circularized DNA
Components 1x rxn 30 x rxn mix
Captured template 2.00 0.00
10uM AmpF6.4Sol 0.50 15.00
10uM AmpR6.3.Ind1 0.50 15.00
2X KAPA SYBR MM 12.50 375.00
H2O 9.50 285.00
Total 25.00
  • Aliquot 23ul, add 2ul of circularized DNA template
Program (Eppendorf Realplex

98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
  • Loaded 3ul of PCR product in TBE gel
File:ZhangLab 2 2013-07-22 19hr 23min TEST-BP-REAL.jpg
  • It seem like I din't see very clear expected bands between 400-480bp, and the background is very high. I wonder if if added too much template, but for previous experiment I add 10ul of template in total 50ul, and got very clear bands.
  • I TEST PCR again by varying template volume (0.5, 1, 2, and 4ul in total 25ul) to test if the volume of template interfere PCR.
  • From the result, it seem like smaller volume of template show less background and stronger expected products. However, it didn't look as clean as I got from the first test experiment. I suspect that Exo I/III may not completely digest ds/ssDNA. I then tested enzyme activity by incubating equal amount of probes and bis-cvt gDNA I used in capture and I saw very clean gel. This suggested that enzymatic activity was good. I also run check circularized template in TBU gel (loaded 3ul) to see if there is the probe or gDNA left. I did not see either. However by PAGE verification, I may not be able to see the left over of probes and gDNA if the level was very low.
File:ZhangLab 2 2013-07-22 15hr 11min confirmExoI-III-digestedprobes.jpg File:ZhangLab 2 2013-07-23 12hr 59min TBU circularized DNA.jpg

Gel1 = ExoI/III with equal amount of probe and bis-cvt gDNA (loaded difeerent amount)
Gel2 = 3ul of NTC and well B5 circularized DNA
  • I decided to used 4% of template and perform PCR amplification in total 100ul reaction to make sequencing libraries.
Components 1x rxn 38 rxn mix
Captured template 4.00 0.00
10uM AmpF6.4Sol 2.00 76.00
10uM AmpR6.3Sol 2.00 0.00
2X KAPA SYBR MM 50.00 1900.00
H2O 42.00 1596.00
Total 100.00
Program (Eppendorf Realplex

98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
  • Purified PCR product with 0.7 volume Ampure beads (I dilute bead 1:1 with 20% PEG/5M NaCl before adding 0.7 volume for purification).
  • Eluted with 30ul H2O
  • Performed PAGE quantification by loading sample 2ul
File:ZhangLab 2 2013-07-23 17hr 11min qPCR BP-REAL A1-H1.jpg File:ZhangLab 2 2013-07-23 17hr 11min qPCR BP-REAL A2-H2.jpg

File:ZhangLab 2 2013-07-23 17hr 59min qPCR BP-REAL A3-H3.jpg File:ZhangLab 2 2013-07-23 17hr 59min qPCR BP-REAL A4-B5.jpg

PAGE quantification[edit]

Well IDs Index Conc. (ng/ul) Yields in 30ul (ng) Well IDs Pool1 volume (ul) Amount Pool2 volume (ul) Amount (ng)
A1 Index 57 6.17 185.16 A1 8.10 50.00 NA NA
B1 Index 58 6.21 186.45 B1 8.05 50.00 NA NA
C1 Index 59 6.42 192.60 C1 7.79 50.00 NA NA
D1 Index 60 6.20 186.10 D1 8.06 50.00 NA NA
E1 Index 61 6.32 189.72 E1 7.91 50.00 NA NA
F1 Index 62 6.61 198.32 F1 7.56 50.00 NA NA
G1 Index 63 6.65 199.44 G1 7.52 50.00 NA NA
H1 Index 64 5.93 177.79 H1 8.44 50.00 NA NA
A2 Index 65 4.94 148.24 A2 10.12 50.00 NA NA
B2 Index 66 5.54 166.12 B2 9.03 50.00 NA NA
C2 Index 67 4.93 147.94 C2 10.14 50.00 NA NA
D2 Index 68 5.56 166.68 D2 9.00 50.00 NA NA
E2 Index 69 4.53 135.81 E2 11.04 50.00 NA NA
F2 Index 70 5.39 161.60 F2 9.28 50.00 NA NA
G2 Index 71 4.99 149.63 G2 10.02 50.00 NA NA
H2 Index 72 5.61 168.28 H2 8.91 50.00 NA NA
A3 Index 73 1.47 44.22 A3 NA NA 12.21 18.00
B3 Index 74 2.11 63.34 B3 23.68 50.00 NA NA
C3 Index 75 2.81 84.45 C3 17.76 50.00 NA NA
D3 Index 76 3.68 110.48 D3 13.58 50.00 NA NA
E3 Index 77 3.92 117.57 E3 12.76 50.00 NA NA
F3 Index 78 3.61 108.40 F3 13.84 50.00 NA NA
G3 Index 79 3.68 110.29 G3 13.60 50.00 NA NA
H3 Index 80 3.67 110.14 H3 13.62 50.00 NA NA
A4 Index 81 0.62 18.67 A4 NA NA 28.93 18.00
B4 Index 82 5.31 159.39 B4 9.41 50.00 NA NA
C4 Index 83 5.10 153.05 C4 9.80 50.00 NA NA
D4 Index 84 5.88 176.34 D4 8.51 50.00 NA NA
E4 Index 85 5.85 175.36 E4 8.55 50.00 NA NA
F4 Index 86 5.89 176.80 F4 8.48 50.00 NA NA
G4 Index 87 5.89 176.78 G4 8.49 50.00 NA NA
H4 Index 88 5.46 163.76 H4 9.16 50.00 NA NA
A5 Index 89 5.24 157.26 A5 9.54 50.00 NA NA
B5 Index 90 4.52 135.63 B5 11.06 50.00 NA NA
  • There were two samples have very low concentration, so I pool these two libraries together and purified in on lane of 5-well TBE gel
  • The rest of libraries were pooled together and purified in 2 of 5-well TBE gels.

PAGE size-selection[edit]

File:ZhangLab 2 2013-07-23 21hr 32min BP-SeqLib-PAGE-SS.jpg File:ZhangLab 2 2013-07-23 21hr 36min BP-SeqLib-PAGE-SS DD-bisPCR e.jpg
  • Pool 1: Library ID: NP-BSPP-BP.norm-Jul19. The libraires were resuspended in 24ul H2O
  • Pool2: Library ID: NP-BSPP-BP.norm_low-Jul19. The libraries were resuspended in 12ul H2O
  • Note: I also perforedm PAGE-size selection of bis PCR performed by Dinh by pooling PCR products normalized based on qt values of qPCR. After PAGE size selection, the libraries were resuspended in 12ul (Library ID: DD-bisPCR-BPrecommended4-Jul19, Index 9-20, 23-44)
  • Alan will do qPCR to quantify concentration of these libraries, and note that DD-bisPCR-BPrecommended4-Jul19 had extramely low concentration.

Sequencing record[edit]

  • Fist Miseq run: MiSeqJul26, 2013

8/01/2013[edit]

  • From sequencing result, seem like we didn't get a good result, and we need to repeat capture.
  • I summarized the total volume/amount/concentration of gDNA samples we received from BP project.
Sample Conc. (ng/ul) Volume for 500ng WellIDs Volume left (ul) Total volume (ul) Total amount (ng) gDNA left gDNA repeat Bis-cvt left bis-cvt repeat Bad qual
1 37.71 13.26 A1 5.80 19.06 718.74 218.74 y 123.00 y
2 39.43 12.68 B1 6.20 18.88 744.46 244.46 y 167.20 yy
3 50.57 9.89 C1 9.50 19.39 980.43 480.43 yy 98.30 y
4 34.86 14.34 D1 5.00 19.34 674.29 174.29 143.80 yy x
5 56.00 8.93 E1 9.50 18.43 1032.00 532.00 yy 117.80 y
6 44.29 11.29 F1 8.00 19.29 854.29 354.29 y 125.60 yy x
7 73.71 6.78 G1 12.00 18.78 1384.57 884.57 yy 136.00 yy
8 29.71 16.83 H1 3.50 20.33 604.00 104.00 138.60 yy
9 40.29 12.41 A2 8.00 20.41 822.29 322.29 y 117.80 y
10 33.43 14.96 B2 12.50 27.46 917.86 417.86 yy 162.00 yy x
11 44.86 11.15 C2 8.50 19.65 881.29 381.29 y 97.78
12 52.86 9.46 D2 10.00 19.46 1028.57 528.57 yy 94.14
13 11.34 9.00 E2 0.00 9.00 102.09 0.00 0.00 x
14 36.86 9.00 F2 0.00 9.00 331.71 0.00 0.00 x
15 26.74 9.00 G2 0.00 9.00 240.69 0.00 0.00
16 50.00 9.00 H2 0.00 9.00 450.00 0.00 10.16 x
17 34.00 14.71 A3 22.00 36.71 1248.00 748.00 yy 0.28 x
18 31.14 16.06 B3 22.00 38.06 1185.14 685.14 yy 9.12 x
19 26.46 18.90 C3 22.00 40.90 1082.06 582.06 yy 0.00 x
20 20.29 20.00 D3 18.50 38.50 781.00 375.29 y 0.00 x
21 18.23 20.00 E3 15.50 35.50 647.11 282.54 y 0.00 x
22 29.43 16.99 F3 22.00 38.99 1147.43 647.43 yy 0.00 x
23 47.71 10.48 G3 22.00 32.48 1549.71 1049.71 yy 31.48
24 32.57 15.35 H3 23.00 38.35 1249.14 749.14 yy 106.36 y
25 29.14 17.16 A4 21.00 38.16 1112.00 612.00 yy 125.60 yy
26 19.60 20.00 B4 18.00 38.00 744.80 352.80 y 149.00 yy x
27 24.31 20.00 C4 19.00 39.00 948.26 461.97 yy 159.40 yy
28 23.66 20.00 D4 19.00 39.00 922.63 449.49 yy 258.20 yy
29 47.43 10.54 E4 9.00 19.54 926.86 426.86 yy 120.40 y x
30 76.86 6.51 F4 12.50 19.01 1460.71 960.71 yy 0.00 x
31 40.00 12.50 G4 7.00 19.50 780.00 280.00 y 461.00 yy
32 32.29 15.49 H4 4.00 19.49 629.14 129.14 578.00 yy x
  • Total amount is the amount of gDNA we got
    • 4 of them (13-16) have total amount < 500ng and we don't have more gDNA/bis-cvt gDNA to repeat experiment. We may need to request more if that's possible and Dr. Zhang agree to do that.
  • gDNA repeat and bis-cvt repeat mean the both gDNA and bis-cvt gDNA are enough to repeat
    • y = lower than required, but expected to be enough
    • yy= equal or more than enough to repeat
  • I am going to do bis-cvt for all samples (except 4 samples) and summarize the list of sample that need to request more if possible.