Ns126:Xliu2014GB: Difference between revisions

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>Shicheng
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====Pair End Mode====
====Pair End Mode====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''N(reads)'''
| align="center" style="background:#f0f0f0;"|'''N(mapped)'''
| align="center" style="background:#f0f0f0;"|'''P(mapping)'''
| align="center" style="background:#f0f0f0;"|'''N(C)'''
| align="center" style="background:#f0f0f0;"|'''N(MCPG)'''
| align="center" style="background:#f0f0f0;"|'''N(MCHG)'''
| align="center" style="background:#f0f0f0;"|'''N(MCHH)'''
| align="center" style="background:#f0f0f0;"|'''N(UCPG)'''
| align="center" style="background:#f0f0f0;"|'''N(UCHG)'''
| align="center" style="background:#f0f0f0;"|'''N(UCHH)'''
| align="center" style="background:#f0f0f0;"|'''P(MCPG)'''
| align="center" style="background:#f0f0f0;"|'''P(MCHG)'''
| align="center" style="background:#f0f0f0;"|'''P(MCHH)'''
|-
| SRR1232302_1_val_1.fq.gz_bismark_bt2_PE_report.txt||10896020||266017||2.40%||10986559||540111||45475||311108||558390||2521979||7009496||49.20%||1.80%||4.20%
|-
| SRR1232303_1_val_1.fq.gz_bismark_bt2_PE_report.txt||20216162||532701||2.60%||21017209||877814||62873||428697||973603||4695560||13978662||47.40%||1.30%||3.00%
|-
| SRR1232304_1_val_1.fq.gz_bismark_bt2_PE_report.txt||9233853||52146||0.60%||2154088||104373||8962||61461||113360||495917||1370015||47.90%||1.80%||4.30%
|-
| SRR1232305_1_val_1.fq.gz_bismark_bt2_PE_report.txt||19402159||359121||1.90%||14853592||750976||48615||303789||702872||3436588||9610752||51.70%||1.40%||3.10%
|-
| SRR1232306_1_val_1.fq.gz_bismark_bt2_PE_report.txt||51400600||1780106||3.50%||71928234||3884870||188607||772020||3360341||16663027||47059369||53.60%||1.10%||1.60%
|-
| SRR1232307_1_val_1.fq.gz_bismark_bt2_PE_report.txt||15015151||255663||1.70%||10806322||480441||44420||298374||509555||2351403||7122129||48.50%||1.90%||4.00%
|-
| SRR1232308_1_val_1.fq.gz_bismark_bt2_PE_report.txt||32508902||278849||0.90%||11147653||519877||35048||231897||507661||2493803||7359367||50.60%||1.40%||3.10%
|-
| SRR1232309_1_val_1.fq.gz_bismark_bt2_PE_report.txt||28578330||270922||0.90%||10742909||496990||27651||125158||386967||2329220||7376923||56.20%||1.20%||1.70%
|-
| SRR1232310_1_val_1.fq.gz_bismark_bt2_PE_report.txt||23879809||240025||1.00%||9580472||431160||36022||285577||493439||2136766||6197508||46.60%||1.70%||4.40%
|-
| SRR1232311_1_val_1.fq.gz_bismark_bt2_PE_report.txt||6078443||57482||0.90%||2159612||91805||6617||49301||91535||469450||1450904||50.10%||1.40%||3.30%
|-
| SRR1232312_1_val_1.fq.gz_bismark_bt2_PE_report.txt||9216625||194894||2.10%||8091546||392407||33197||226776||428295||1871966||5138905||47.80%||1.70%||4.20%
|-
|
|}


== Bam to Methylfreq==
== Bam to Methylfreq==

Revision as of 19:12, 21 January 2016

Background

  • Construction protocol: Genomic DNA from the tumor and corresponding adjacent tissues was prepared using the QIAamp DNA Blood Mini Kit(Qiagen) following the manufacturer’s instructions. Prior to the library construction, 3µg of genomic DNA from each sample was fragmented using a Covarias sonication system to mean sizes of approximately 200-300bp. After fragmentation, libraries were constructed according to the Illumina Paired-End protocol. Briefly, the purified, randomly fragmented DNA was treated with a mix of T4 DNA polymerase, Klenow fragments, T4 polynucleotide kinase and a nucleotide triphosphate mix to repair the ends by blunting and phosphorylation. The blunted DNA fragments were subsequently 3’-adenylated using the Klenow fragment (3’-5’exo) and ligated by T4 DNA ligase to adapters synthesized with 5’ –methyl-cytosine instead of cytosine. The adaptor-ligated library was purified using AMPure XP beads (Beckman Coulter Genomics). 500 ng of each library was hybridized to Agilent SureSelect Methyl-Seq biotinylated RNA baits (84 Mb) for 24 h at 65 C. Biotinylated target hybrids were captured on Dynal MyOne Streptavidin T1 (Invitrogen), and purified through MinElute PCR column (Qiagen). Bisulfite conversion of the purified captured library was performed using the EZ DNA Methylation Gold Kit (Zymo Research) as per manufacturer’s instructions. The bisulfite converted captured library was amplified by PCR with 14 PCR cycles, then purified by AMPure XP beads and quantified by Agilent 2100 system and qPCR. Methyl-Seq libraries were then sequenced on the HiSeq 2000 platform according to the manufacturer’s instructions, and 2 ×90bp paired-end reads were generated. Methyl-Seq libraries were prepared for sequencing using standard Illumina protocols.
  • We propose a statistical algorithm MethylPurify that uses regions with bisulfite reads showing discordant methylation levels to infer tumor purity from tumor samples alone. With purity estimate, MethylPurify can identify differentially methylated regions (DMRs) from individual tumor samples without genomic variation information or prior knowledge from other datasets. In simulations with mixed bisulfite reads from cancer and normal cell lines, MethylPurify correctly inferred tumor purity and identified over 96% of the DMRs. On real patient data where tumor to normal comparison were used as golden standard, MethylPurify called DMR from tumor samples alone at over 57% sensitivity and 91% specificity.
  • Lung adenocarcinoma cancer and normal tissues from 5 patients were captured by Agilent SureSelect Methyl-Seq system, followed by bisulfite sequencing.

Method

Sample

title geo_accession channel_count tissue patient id smoking Gender Alignment processing sequencing library relation relation relation
75A-2 GSM1367123 1 adjacent nomal lung tissue 75 smoker male bsmap MethylPurify HiSeq 2000 Bisulfite-Seq SAMN02725476 SRX515122 SRR1232302
75B GSM1367124 1 lung adenocarcinoma tissue 75 smoker male bsmap MethylPurify HiSeq 2001 Bisulfite-Seq SAMN02725471 SRX515123 SRR1232303
109A-2 GSM1367125 1 adjacent nomal lung tissue 109 non-smoker male bsmap MethylPurify HiSeq 2002 Bisulfite-Seq SAMN02725468 SRX515124 SRR1232304
109B GSM1367126 1 lung adenocarcinoma tissue 109 non-smoker male bsmap MethylPurify HiSeq 2003 Bisulfite-Seq SAMN02725475 SRX515125 SRR1232305
109C GSM1367127 1 corresponding lymph node metastasis tissue 109 non-smoker male bsmap MethylPurify HiSeq 2004 Bisulfite-Seq SAMN02725470 SRX515126 SRR1232306
137A GSM1367128 1 adjacent nomal lung tissue 137 non-smoker female bsmap MethylPurify HiSeq 2005 Bisulfite-Seq SAMN02725467 SRX515127 SRR1232307
137B GSM1367129 1 lung adenocarcinoma tissue 137 non-smoker female bsmap MethylPurify HiSeq 2006 Bisulfite-Seq SAMN02725477 SRX515128 SRR1232308
137C GSM1367130 1 corresponding lymph node metastasis tissue 137 non-smoker female bsmap MethylPurify HiSeq 2007 Bisulfite-Seq SAMN02725469 SRX515129 SRR1232309
156A-2 GSM1367131 1 adjacent nomal lung tissue 156 non-smoker female bsmap MethylPurify HiSeq 2008 Bisulfite-Seq SAMN02725472 SRX515130 SRR1232310
156B-2 GSM1367132 1 lung adenocarcinoma tissue 156 non-smoker female bsmap MethylPurify HiSeq 2009 Bisulfite-Seq SAMN02725478 SRX515131 SRR1232311
201A GSM1367133 1 adjacent nomal lung tissue 201 non-smoker female bsmap MethylPurify HiSeq 2010 Bisulfite-Seq SAMN02725474 SRX515132 SRR1232312
201B GSM1367134 1 lung adenocarcinoma tissue 201 non-smoker female bsmap MethylPurify HiSeq 2011 Bisulfite-Seq SAMN02725473 SRX515133 SRR1232313

Sample Matching Table

SRR_Normal SRR_Cancer patient id smoking Gender
SRR1232302 SRR1232303 75 smoker male
SRR1232304 SRR1232305 109 non-smoker male
SRR1232307 SRR1232308 137 non-smoker female
SRR1232310 SRR1232311 156 non-smoker female
SRR1232312 SRR1232313 201 non-smoker female

SRA Download

  • sra saved in: /home/shg047/ncbi/public/sra
prefetch -v SRR1232302 &
prefetch -v SRR1232303 &
prefetch -v SRR1232304 &
prefetch -v SRR1232305 &
prefetch -v SRR1232306 &
prefetch -v SRR1232307 &
prefetch -v SRR1232308 &
prefetch -v SRR1232309 &
prefetch -v SRR1232310 &
prefetch -v SRR1232311 &
prefetch -v SRR1232312 &
prefetch -v SRR1232313 &

SRA to Fastq

fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232302.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232303.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232304.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232305.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232306.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232307.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232308.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232309.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232310.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232311.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232312.sra &
fastq-dump --outdir /home/shg047/oasis/Xliu2014 --split-files /home/shg047/ncbi/public/sra/SRR1232313.sra &

Fastq to Bam

trim_glore

#!/bin/csh
#PBS -q glean
#PBS -l nodes=1:ppn=1
#PBS -l walltime=6:00:00
#PBS -o SRR1232309_2.log
#PBS -e SRR1232309_2.err
#PBS -V
#PBS -M shihcheng.guo@gmail.com
#PBS -m abe
#PBS -A k4zhang-group
cd /oasis/tscc/scratch/shg047/Xliu2014/fastq
gzip SRR1232309_2.fastq
trim_galore --phred33 --fastqc --illumina SRR1232309_2.fastq --output_dir ../fastq_trim

Alignment

#!/bin/csh
#PBS -n Xie.LUN.SRR1232310_1.fastq.gz
#PBS -q glean
#PBS -l nodes=1:ppn=1
#PBS -l walltime=72:00:00
#PBS -o SRR1232310_1.fastq.gz.trim.log
#PBS -e SRR1232310_1.fastq.gz.trim.err
#PBS -V
#PBS -M shihcheng.guo@gmail.com
#PBS -m abe
#PBS -A k4zhang-group
cd /oasis/tscc/scratch/shg047/Xliu2014/fastq
bismark --bowtie2 --non_directional --phred33-quals --fastq -L 30 -N 1 -s 10 --multicore 6 /home/shg047/db/hg19/meth/bismark -1 ../fastq2_trim/SRR1232310_1_val_1.fq.gz -2 ../fastq2_trim/SRR1232310_2.fastq.gz_val_2.fq.gz  -o ../bam2
  • check the fastqc and find the first 10 base were not suitable to be aligned in the mapping.

Alignment

Single End Mode

Sample N(reads) N(mapped) P(mapping) N(C) N(MCPG) N(MCHG) N(MCHH) N(UCPG) N(UCHG) N(UCHH) P(MCPG) P(MCHG) P(MCHH)
SRR1232302_1 10901797 1965533 18.00% 42473403 2015096 267778 2154224 2124299 9385818 26526188 48.70% 2.80% 7.50%
SRR1232302_2 10896097 5418 0.00% 30335 277 270 17333 80 440 11935 77.60% 38.00% 59.20%
SRR1232303_1 20216456 3779429 18.70% 81622770 3481428 366020 2832465 4272866 18062973 52607018 44.90% 2.00% 5.10%
SRR1232303_2 20227713 9178 0.00% 54677 475 527 24588 345 1090 27652 57.90% 32.60% 47.10%
SRR1232304_1 11083547 2036357 18.40% 44074525 2056785 273185 2182017 2309149 9831347 27422042 47.10% 2.70% 7.40%
SRR1232304_2 11080702 4983 0.00% 30242 268 258 16560 157 811 12188 63.10% 24.10% 57.60%
SRR1232305_1 19402409 3165722 16.30% 67606037 3274247 330762 2419983 3246336 15086910 43247799 50.20% 2.10% 5.30%
SRR1232305_2 19480905 6045 0.00% 39894 475 329 16448 261 1473 20908 64.50% 18.30% 44.00%
SRR1232306_1 51509964 4127537 8.00% 80772795 4545044 177613 927450 4234671 19085576 51802441 51.80% 0.90% 1.80%
SRR1232306_2 51400718 14462 0.00% 49976 1073 287 15465 551 2003 30597 66.10% 12.50% 33.60%
SRR1232307_1 15046156 2156610 14.30% 46309778 2026003 289597 2342114 2157847 10053826 29440391 48.40% 2.80% 7.40%
SRR1232307_2 15015245 10801 0.10% 54587 489 1109 34079 84 498 18328 85.30% 69.00% 65.00%
SRR1232308_1 35592158 4877978 13.70% 105500558 4855743 475644 3675162 5068193 23245581 68180235 48.90% 2.00% 5.10%
SRR1232308_2 35440375 20675 0.10% 113086 1092 2036 51007 490 1665 56796 69.00% 55.00% 47.30%
SRR1232309_1 34522244 2000970 5.80% 38231876 1945505 83009 459587 1653844 8673938 25415993 54.10% 0.90% 1.80%
SRR1232309_2 34294088 20558 0.10% 53308 1018 778 15401 201 756 35154 83.50% 50.70% 30.50%
SRR1232310_1 24123644 2324975 9.60% 45940356 2011191 269884 2612542 2362908 9839244 28844587 46.00% 2.70% 8.30%
SRR1232310_2 23879911 10879 0.00% 64230 733 557 34990 346 1483 26121 67.90% 27.30% 57.30%
SRR1232311_1 12239701 1250928 10.20% 24422478 1035635 113669 985226 1171198 5200394 15916356 46.90% 2.10% 5.80%
SRR1232311_2 12156959 5373 0.00% 36113 317 485 16080 149 720 18362 68.00% 40.20% 46.70%
SRR1232312_1 9216954 1635472 17.70% 35503016 1655556 221162 1785193 1890919 7936466 22013720 46.70% 2.70% 7.50%
SRR1232312_2 9221469 5019 0.10% 28390 137 259 16089 19 165 11721 87.80% 61.10% 57.90%
SRR1232313_2 32789205 16897 0.10% 100383 441 749 46445 117 511 52120 79.00% 59.40% 47.10%

Pair End Mode

Sample N(reads) N(mapped) P(mapping) N(C) N(MCPG) N(MCHG) N(MCHH) N(UCPG) N(UCHG) N(UCHH) P(MCPG) P(MCHG) P(MCHH)
SRR1232302_1_val_1.fq.gz_bismark_bt2_PE_report.txt 10896020 266017 2.40% 10986559 540111 45475 311108 558390 2521979 7009496 49.20% 1.80% 4.20%
SRR1232303_1_val_1.fq.gz_bismark_bt2_PE_report.txt 20216162 532701 2.60% 21017209 877814 62873 428697 973603 4695560 13978662 47.40% 1.30% 3.00%
SRR1232304_1_val_1.fq.gz_bismark_bt2_PE_report.txt 9233853 52146 0.60% 2154088 104373 8962 61461 113360 495917 1370015 47.90% 1.80% 4.30%
SRR1232305_1_val_1.fq.gz_bismark_bt2_PE_report.txt 19402159 359121 1.90% 14853592 750976 48615 303789 702872 3436588 9610752 51.70% 1.40% 3.10%
SRR1232306_1_val_1.fq.gz_bismark_bt2_PE_report.txt 51400600 1780106 3.50% 71928234 3884870 188607 772020 3360341 16663027 47059369 53.60% 1.10% 1.60%
SRR1232307_1_val_1.fq.gz_bismark_bt2_PE_report.txt 15015151 255663 1.70% 10806322 480441 44420 298374 509555 2351403 7122129 48.50% 1.90% 4.00%
SRR1232308_1_val_1.fq.gz_bismark_bt2_PE_report.txt 32508902 278849 0.90% 11147653 519877 35048 231897 507661 2493803 7359367 50.60% 1.40% 3.10%
SRR1232309_1_val_1.fq.gz_bismark_bt2_PE_report.txt 28578330 270922 0.90% 10742909 496990 27651 125158 386967 2329220 7376923 56.20% 1.20% 1.70%
SRR1232310_1_val_1.fq.gz_bismark_bt2_PE_report.txt 23879809 240025 1.00% 9580472 431160 36022 285577 493439 2136766 6197508 46.60% 1.70% 4.40%
SRR1232311_1_val_1.fq.gz_bismark_bt2_PE_report.txt 6078443 57482 0.90% 2159612 91805 6617 49301 91535 469450 1450904 50.10% 1.40% 3.30%
SRR1232312_1_val_1.fq.gz_bismark_bt2_PE_report.txt 9216625 194894 2.10% 8091546 392407 33197 226776 428295 1871966 5138905 47.80% 1.70% 4.20%

Bam to Methylfreq

MethylKit

MethylPurify