Alice:LabNotes/2010-3-8: Difference between revisions

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  95C 3min -> (95C 3sec -> 60C 20sec -> 72C 3sec) x40 -> 72C 3min -> 15C hold
  95C 3min -> (95C 3sec -> 60C 20sec -> 72C 3sec) x40 -> 72C 3min -> 15C hold


==Result==
===results===
 
===conclusion===
*It seems like we were able to capture regions covered on chr7, and got enrichment level that is 2 to 8 fold less than the gDNA across different samples.
*It seems like we were able to capture regions covered on chr7, and got enrichment level that is 2 to 8 fold less than the gDNA across different samples.


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===results===
===results===
*From the above results, we can conclude that commercialized Nimblegen kits definitely had the best enrichment level out of all. However, our padlock probe captured libraries also had satisfactory level of enrichment across several different regions of chromosomes. Also, it seems like we can skip the wash step as suggested by the results from past few experiments.
 
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===conclusion===
*From the above results, we can conclude that commercialized Nimblegen kits definitely had the best enrichment level out of all. However, our padlock probe captured libraries also had satisfactory level of enrichment across several different regions of chromosomes. Also, it seems like we can skip the wash step as suggested by the results from past few experiments.

Revision as of 20:26, 18 March 2010

Prepare genomic DNA for hybridization

  • samples to be prepared:
  • sample 1: CVF-gDNA
  • sample 2: CViB-gDNA

two sheared gDNA received from Harvard:

  • sample 3: CD1 PGP1 ips P16 (30ul)
  • sample 4: PGP1F 8 gDNA (30ul)

End-repair Reactions

Fragmented DNA                             85 ul   30ul for sample 3,4
10X End Repair Reaction Buffer             10 ul   4ul for sample 3,4
End Repair Enzyme Mix                      5  ul   5ul for sample 3,4
Keep the tube at room temperature (~20°C) for 30 minutes.
Purify with Qiaquick column and elute in 40ul ddH2O
Note: for blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the
chance of getting chimeric reads.
  • nanodrop result:
  • CVf: 45ng/ul *40ul
  • CD1 PGP1 ips P16: 137.8ng/ul
  • PGP1F 8 gDNA: 146.3ng/ul * 40ul

A-Tailing reactions

Blunt-end DNA                             40 ul
10X dA-Tailing Reaction Buffer (10X)       5 ul
Klenow Fragment (3’-5’ exo-)               3 ul
Incubated at 37C for 30min
purified the products with Qiaquick column and elute in 40ul ddH2O

adaptor ligation

Prepare adaptors (need to be done only for the first time): 
100uM PE-t: 20ul
100uM PE-b: 20ul
10x stoffel buffer:  10ul
H2O:        50ul
94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec. 
commonly used adaptors:
Blunt-end adaptors:
5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH	 	Solexa_1_up
3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH	        Solexa_1_lo_nop

TA adaptors (for the one adaptor protocol):
5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH		PE_t_adapter
3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos	                t_adaptor_rc_s

regular Y adaptor:
PE_t_adaptor(top)              ACACTCTTTCCCTACACGACGCTCTTCCGATC*T              3'-Phosphorothioate bond	
PE_b_adaptor(bottom)           \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG       5'-phosphorylation	
Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw 
cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. 
adaptor:target molar ratio is 1:10~20
A-tailed DNA                               10 ul for CVF, 3ul for CViB, CD1 PGP1 ips, and PGP1F 8
20uM Y adaptor                             3 ul 
5X Quick ligase buffer                     10 ul  
Quick Ligase                                3 ul
H2O                                        24 ul for CVF, 31ul for CViB, CD1 PGP1 ips, and PGP1F 8                                      
Incubate at room temperature for 15 minutes
purify the product with Agencourt AMpure kit and elute in 40ul ddH2O

PCR

Ligation products                 5ul      (2 well for each set, total of 8 well) 
100uM Solexa_PCR_up               0.2ul        
100uM solexa_PCR-lo               0.2ul
2X phusion HF master mix          50ul     
50X SYBR Green I                  0.4ul      
H2O                               45ul       
PCR program: 98 °C 30sec  -> 13 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. 
purify the products with Qiagen Qiaquick columns and elute in 40ul ddH2O

Biotin-labeled probe capture

  • gDNA samples of DF and foreskin will be used, and the preparation protocol can be found under labnote 2-1-2010

working buffer preparation

1x binding buffer (following Nimblegen setup):
1M NaCl: 1000ul
10mM Tris-HCl (pH ~7): 25ul
1mM EDTA: 5ul
ddH2O: 1470ul
total: 2.5ml

Hybrid selection

Mix the following to 20ul total volume: 
1ug ligated DNA with 200ng padlock probes
2.5ug of human Cot-1 DNA
2ul of 10X AmpLigase buffer
1ul of 100uM competing oligos each (Solexa_up and Solexa_lo)
[gDNA]
DF6-9-9: 221ng/ul (5ul needed)
foreskin: 153ng/ul (6.5ul needed)
exome probe set 1: w/o PAGE selection (with Jan09 1-5,8,9,Mar R2, R3): 7ng/ul (28ul needed)
exome probe set 2: w/ PAGE selection (Jan09 1-5,8,9): 7ng/ul (28ul)

system setup (all units are in ul):

reagents DF DF(for exome probe set2) foreskin
DNA 5 5 6.5
probe 28 28 28
10x ampligase buffer 5 5 5
human Cot-1 DNA 2.5 2.5 2.5
Solexa_up (100uM) 1 1 1
Solexa_lo (100uM) 1 1 1
ddH2O 6 6 4.5
total: 50 50 50
95C 5min -> cool down to 60C at 0.01C/sec -> 60C 24 hours -> 50C 24 hours.

Prepare the Streptavidin Dynabeads

a. Take 50ul M-280 streptavidin Dynalbeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear
b. add twice the volume of the beads of 1x binding buffer, place the tube on magnet and remove the liquid when the solution becomes clear
c. wash the beads for a total of  3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA)
d. resuspend the beads in 200ul 1x binding buffer, warm up to 45C. 
c. Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min, take 1/2 and go on with no wash. 
d. Remove the liquid from the beads with a magnet.
e. Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 45C.
f. After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min.
g. Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5. 
h. Purifythe 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB.

Post-capture PCR (done on 3/15/10)

a.Set up the reaction system with Phusion High-Fidelity PCR master mix:
  2x Phusion master mix:     50ul
  100uM PCR_F		     0.5ul
  100uM PCR_R		     0.5ul
  50X SYBG I		     0.8ul
  Captured DNA		     15ul
  H2O 			     44ul
b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. 
First batch of samples used 20 cycles. For second batch of samples, DF N.W. and DF #2 N.W. only used 15 cycles, while the other had 19 cycles.
Terminate the reaction when the amplification curves approach to the plateau.
purify the products with Qiaquick columns and elute in 30ul EB
Nanodrop result:
DF N.W:         177.4ng/ul
DF set#2 45C:   34.7ng/ul
DF set#2 N.W.:  208.8ng/ul
DF 45C:         141ng/ul
foreskin 45C:   158.6ng/ul
foreskin N.W.:  224.8ng/ul

PAGE gel image:
File:ZhangLab 2 2010-03-15 16hr 33min.jpg File:ZhangLab 2 2010-03-15 16hr 36min.jpg

Measurement of Enrichment using qPCR on biotin-labeled probe capture sample

  • The qPCR assay is used to estimate relative fold-enrichment by measuring the relative abundance of control targets in amplified sample library and amplified captured DNA to determine whether the capture was successful.

Preparation of reagents

NSC qPCR assay name primer sequence 5'->3' Tm ( C) length
NSC-0237 F: CGCATTCCTCATCCCAGTATG 81.15 80bp
R: AAAGGACTTGGTGCAGAGTTCAG
NSC-0247 F: CCCACCGCCTTCGACAT 81.03 74bp
R: CCTGCTTACTGTGGGCTCTTG
NSC-0268 F: CTCGCTTAACCAGACTCATCTACTGT 78.99 75bp
R: ACTTGGCTCAGCTGTATGAAGGT
NSC-0272 F: CAGCCCCAGCTCAGGTACAG 82.23 71bp
R: ATGATGCGAGTGCTGATGATG
  • These are the primers that I have designed to test the enrichment level of my probes
qPCR assay name chromosome position primer sequence 5'->3'
QPCR_chr5_L chr5:176328156-176328295 CCCTTCTCTGAAAAGCTCCT
QPCR_chr5_R TGCAGTGATACAAGAACATGC
QPCR_chr7_L chr7:77048663-77048795 GGCAAGAAAAGAGGTGAAGC
QPCR_chr7_R GCACCAGATAGCTAGACCAG
QPCR_chr11_L chr11:5925385-5925520 TTGGGATGGTTGCCTTTTTG
QPCR_chr11_R GCCATTCCATGGTGGTAAGT
QPCR_chr10_L chr10:17952266-17952400 GCCGGAGTAGTCATCATTGT
QPCR_chr10_R TAGGTGCACACGTCTTTTCT
  1. Dilute the NSC assay forward and reverse primers to 2μM.
  2. Dilute sufficient amounts of amplified sample library and amplified captured DNA to a concentration of 5ng/μl in PCR grade water for use as qPCR templates

PCR reaction setup

                                              x6    x3(3 primers:chr5,7,11)
PCR grade water                   19.7ul
Primer R+F(2uM each)              2ul
template (5ng/ul)                 3.3ul
KAPA 2x master mix                25ul
95C 3min -> (95C 3sec -> 60C 20sec -> 72C 3sec) x40 -> 72C 3min -> 15C hold

results

conclusion

  • It seems like we were able to capture regions covered on chr7, and got enrichment level that is 2 to 8 fold less than the gDNA across different samples.
  • For DF set2 with 45C wash, I didn't get good amount of amplification during the last PCR step, I would think this is somewhat related to the low/zero enrichment level we seen in the graph. However, this is inconsistent with our previous results. Actually, in last enrichment analysis PCR, I found a mistake. I used SYBR green along with Kapa master mix. Kapa already included SYBR green, therefore adding extra SYBR green should be problematic (high concentration causes inhibition), thus the results from last time wasn't too trustworthy.
  • On chr11, I saw slight rise in the curve toward very late stage of PCR on the gDNA samples. It would be good to repeat another QPCR with 50 cycles or so to see whether we will get anything useful. I am planning to do another QPCR analysis to test chr10, four NSC primers, and repeat chr7 on different 6 samples(since PCR is limited to 96 wells).

2nd PCR reaction setup (done on 3/16/10)

                                             x8    x6(6 primers:4 NSC, chr7,10)     
PCR grade water                   19.7ul
Primer R+F(2uM each)              2ul
template (5ng/ul)                 3.3ul
Iq SYBR green 2x master mix       25ul
95C 3min -> (95C 15sec -> 60C 20sec -> 72C 30sec) x40 -> 72C 3min -> 15C hold

results

Well / Set Content sample ID Efficiency C(t) d1=Sample C(t) – gDNA C(t) enrichment ratio (2^d1) mean ' d2=sample C(t) – Nimblegen data C(t) enrichment ratio (2^d2) mean
A1 Sample DF-no wash-Chr11 34.84% 28.27 3.92 15.14 16.7 8.67 407.31 3877.94
A2 Sample DF-no wash-Chr11 33.29% 28.14 3.35 10.2 8.17 288.01
A3 Sample DF-no wash-Chr7 42.42% 26.18 3.76 13.55 -0.92 0.53
A4 Sample DF-no wash-Chr7 52.80% 25.93 3.34 10.13 0.35 1.27
A5 Sample DF-no wash-NSC 237 44.30% 27.89 3.18 9.06 11.28 2486.67
A6 Sample DF-no wash-NSC 237 37.19% 28.69 2.42 5.35 12.65 6427.31
A7 Sample DF-no wash-NSC 268 44.07% 26.56 2.75 6.73 11.41 2721.15
A8 Sample DF-no wash-NSC 268 46.93% 26.55 1.84 3.58 11.76 3468.27
A9 Sample DF-no wash-NSC 247 19.11% 28.86 5 32 13.05 8480.89
A10 Sample DF-no wash-NSC 247 17.82% 30.36 6.36 82.14 14.05 16961.78
A11 Sample DF-no wash-NSC 272 27.54% 29.12 3.19 9.13 11.74 3420.52
A12 Sample DF-no wash-NSC 272 35.59% 28.58 1.79 3.46 10.87 1871.53
B1 Sample DF set2-no wash-Chr11 34.16% 28.03 3.68 12.82 6.42 8.43 344.89 1561.74
B2 Sample DF set2-no wash-Chr11 40.07% 27.99 3.2 9.19 8.02 259.57
B3 Sample DF set2-no wash-Chr7 50.85% 25.66 3.24 9.45 -1.44 0.37
B4 Sample DF set2-no wash-Chr7 40.60% 26 3.41 10.63 0.42 1.34
B5 Sample DF set2-no wash-NSC 237 37.64% 28.21 3.5 11.31 11.6 3104.19
B6 Sample DF set2-no wash-NSC 237 40.56% 28.68 2.41 5.31 12.64 6382.92
B7 Sample DF set2-no wash-NSC 268 42.94% 26.13 2.32 4.99 10.98 2019.8
B8 Sample DF set2-no wash-NSC 268 42.47% 26.31 1.6 3.03 11.52 2936.74
B9 Sample DF set2-no wash-NSC 247 44.94% 24.9 1.04 2.06 9.09 544.96
B10 Sample DF set2-no wash-NSC 247 35.02% 25.15 1.15 2.22 8.84 458.25
B11 Sample DF set2-no wash-NSC 272 44.63% 27.69 1.76 3.39 10.31 1269.46
B12 Sample DF set2-no wash-NSC 272 34.67% 28.18 1.39 2.62 10.47 1418.35
C1 Sample DF-45C wash-Chr11 34.35% 28.12 3.77 13.64 7.25 8.52 367.09 2058.92
C2 Sample DF-45C wash-Chr11 33.56% 28.31 3.52 11.47 8.34 324.03
C3 Sample DF-45C wash-Chr7 38.31% 25.09 2.67 6.36 -2.01 0.25
C4 Sample DF-45C wash-Chr7 49.62% 24.74 2.15 4.44 -0.84 0.56
C5 Sample DF-45C wash-NSC 237 39.28% 28.27 3.56 11.79 11.66 3236.01
C6 Sample DF-45C wash-NSC 237 39.49% 28.23 1.96 3.89 12.19 4672.57
C7 Sample DF-45C wash-NSC 268 49.68% 26.87 3.06 8.34 11.72 3373.43
C8 Sample DF-45C wash-NSC 268 51.21% 26.8 2.09 4.26 12.01 4124.49
C9 Sample DF-45C wash-NSC 247 40.09% 25.39 1.53 2.89 9.58 765.36
C10 Sample DF-45C wash-NSC 247 40.44% 25.79 1.79 3.46 9.48 714.11
C11 Sample DF-45C wash-NSC 272 38.29% 29.37 3.44 10.85 11.99 4067.71
C12 Sample DF-45C wash-NSC 272 38.71% 29.29 2.5 5.66 11.58 3061.45
E1 Sample foreskin-no wash-chr 10 43.11% 26.85 2.5 5.66 4.64
E2 Sample foreskin-no wash-chr 10 41.44% 26.97 2.18 4.53
E3 Sample foreskin-no wash-chr 7 48.40% 25.36 2.94 7.67
E4 Sample foreskin-no wash-chr 7 48.11% 25.59 3 8
E5 Sample foreskin-no wash-NSC 237 45.61% 27.89 3.18 9.06
E6 Sample foreskin-no wash-NSC 237 36.37% 28.6 2.33 5.03
E7 Sample foreskin-no wash-NSC 268 40.52% 26.08 2.27 4.82
E8 Sample foreskin-no wash-NSC 268 41.44% 26.2 1.49 2.81
E9 Sample foreskin-no wash-NSC 247 45.23% 24.67 0.81 1.75
E10 Sample foreskin-no wash-NSC 247 37.46% 25.16 1.16 2.23
E11 Sample foreskin-no wash-NSC 272 43.49% 27.15 1.22 2.33
E12 Sample foreskin-no wash-NSC 272 41.18% 27.64 0.85 1.8
F1 Sample foreskin-45C-chr 10 40.86% 26.6 2.25 4.76 7.23
F2 Sample foreskin-45C-chr 10 41.86% 26.66 1.87 3.66
F3 Sample foreskin-45C-chr 7 44.72% 25.26 2.84 7.16
F4 Sample foreskin-45C-chr 7 44.65% 25.2 2.61 6.11
F5 Sample foreskin-45C-NSC 237 43.36% 28.64 3.93 15.24
F6 Sample foreskin-45C-NSC 237 33.89% 29.73 3.46 11
F7 Sample foreskin-45C-NSC 268 49.90% 25.96 2.15 4.44
F8 Sample foreskin-45C-NSC 268 41.19% 26.18 1.47 2.77
F9 Sample foreskin-45C-NSC 247 43.75% 26.49 2.63 6.19
F10 Sample foreskin-45C-NSC 247 35.44% 27.08 3.08 8.46
F11 Sample foreskin-45C-NSC 272 44.04% 29.03 3.1 8.57
F12 Sample foreskin-45C-NSC 272 43.25% 29.86 3.07 8.4
G1 Standard foreskin-gDNA-chr10 27.51% 24.35
G2 Standard foreskin-gDNA-chr10 29.77% 24.79
G3 Standard foreskin-gDNA-chr7 36.64% 22.42
G4 Standard foreskin-gDNA-chr7 38.12% 22.59
G5 Standard foreskin-gDNA-NSC 237 33.36% 24.71
G6 Standard foreskin-gDNA-NSC 237 24.24% 26.27
G7 Standard foreskin-gDNA-NSC 268 37.41% 23.81
G8 Standard foreskin-gDNA-NSC 268 28.89% 24.71
G9 Standard foreskin-gDNA-NSC 247 34.97% 23.86
G10 Standard foreskin-gDNA-NSC 247 27.15% 24
G11 Standard foreskin-gDNA-NSC 272 37.33% 25.93
G12 Standard foreskin-gDNA-NSC 272 30.36% 26.79
H1 Sample DF-nimblegen-chr10 37.15% 19.6
H2 Sample DF-nimblegen-chr10 39.72% 19.97
H3 Sample DF-nimblegen-chr7 23.10% 27.1
H4 Sample DF-nimblegen-chr7 34.14% 25.58
H5 Sample DF-nimblegen-NSC 237 39.28% 16.61
H6 Sample DF-nimblegen-NSC 237 33.44% 16.04
H7 Sample DF-nimblegen-NSC 268 35.90% 15.15
H8 Sample DF-nimblegen-NSC 268 41.22% 14.79
H9 Sample DF-nimblegen-NSC 247 41.80% 15.81
H10 Sample DF-nimblegen-NSC 247 34.62% 16.31
H11 Sample DF-nimblegen-NSC 272 37.82% 17.38
H12 Sample DF-nimblegen-NSC 272 38.79% 17.71

conclusion

  • From the above results, we can conclude that commercialized Nimblegen kits definitely had the best enrichment level out of all. However, our padlock probe captured libraries also had satisfactory level of enrichment across several different regions of chromosomes. Also, it seems like we can skip the wash step as suggested by the results from past few experiments.