Ns126:Xliu2014GB: Difference between revisions
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Output specified: comprehensive | Output specified: comprehensive | ||
No overlapping methylation calls specified | No overlapping methylation calls specified | ||
Collect 0-based Coverage File: | |||
== Bam to SortBam (Feb 18 2016 Finished)== | == Bam to SortBam (Feb 18 2016 Finished)== |
Revision as of 19:16, 24 February 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.
- Zheng X, Zhao Q, Wu HJ, Li W et al. MethylPurify: tumor purity deconvolution and differential methylation detection from single tumor DNA methylomes. Genome Biol 2014 Aug 7;15(8):419. PMID: 25103624
- It is interesting, Dinh also repeat the analysis before and her wiki: http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/Dinh_2014/NOTES/2014-9-15
Method
- http://www.ncbi.nlm.nih.gov/sra/SRX515122[accn]
- http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56712
- Methypurity: Install and Usage
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 --clip_R1 10 --clip_R2 10 --three_prime_clip_R1 5 --three_prime_clip_R1 5 SRR1232309_2.fastq --output_dir ../fastq_trim
Sample | N(reads) | P(adaptor) | P(pass1) | Base(filting) | PB(pass2) |
SRR1232302_1.fastq.gz_trimming_report.txt | 10901814 | 34.00% | 100.00% | 2.10% | 97.20% |
SRR1232302_2.fastq.gz_trimming_report.txt | 10896296 | 55.10% | 100.00% | 1.80% | 97.40% |
SRR1232303_1.fastq.gz_trimming_report.txt | 20216497 | 34.20% | 100.00% | 2.00% | 97.40% |
SRR1232303_2.fastq.gz_trimming_report.txt | 20227949 | 54.60% | 100.00% | 1.70% | 97.50% |
SRR1232304_1.fastq.gz_trimming_report.txt | 11083565 | 32.40% | 100.00% | 2.40% | 97.00% |
SRR1232304_2.fastq.gz_trimming_report.txt | 11080866 | 54.30% | 100.00% | 2.10% | 97.20% |
SRR1232305_1.fastq.gz_trimming_report.txt | 19402456 | 36.20% | 100.00% | 1.80% | 97.50% |
SRR1232305_2.fastq.gz_trimming_report.txt | 19481098 | 54.80% | 100.00% | 1.60% | 97.60% |
SRR1232306_1.fastq.gz_trimming_report.txt | 51510022 | 32.10% | 100.00% | 2.00% | 97.30% |
SRR1232306_2.fastq.gz_trimming_report.txt | 51401414 | 52.70% | 100.00% | 1.80% | 97.50% |
SRR1232307_1.fastq.gz_trimming_report.txt | 15046190 | 37.80% | 100.00% | 2.60% | 96.40% |
SRR1232307_2.fastq.gz_trimming_report.txt | 15015457 | 60.50% | 100.00% | 2.30% | 96.50% |
SRR1232308_1.fastq.gz_trimming_report.txt | 35592228 | 40.50% | 100.00% | 2.40% | 96.50% |
SRR1232308_2.fastq.gz_trimming_report.txt | 35440880 | 61.50% | 100.00% | 2.20% | 96.60% |
SRR1232309_1.fastq.gz_trimming_report.txt | 34522276 | 36.20% | 100.00% | 2.60% | 96.40% |
SRR1232309_2.fastq.gz_trimming_report.txt | 34294567 | 59.10% | 100.00% | 2.40% | 96.50% |
SRR1232310_1.fastq.gz_trimming_report.txt | 24123686 | 28.40% | 100.00% | 17.70% | 81.70% |
SRR1232310_2.fastq.gz_trimming_report.txt | 23880345 | 54.90% | 100.00% | 2.40% | 96.90% |
SRR1232311_1.fastq.gz_trimming_report.txt | 12239714 | 28.80% | 100.00% | 17.20% | 82.30% |
SRR1232311_2.fastq.gz_trimming_report.txt | 12157130 | 54.70% | 100.00% | 2.20% | 97.00% |
SRR1232312_1.fastq.gz_trimming_report.txt | 9216970 | 33.50% | 100.00% | 2.60% | 96.70% |
SRR1232312_2.fastq.gz_trimming_report.txt | 9221739 | 56.30% | 100.00% | 2.60% | 96.60% |
SRR1232313_1.fastq.gz_trimming_report.txt | 32811085 | 34.60% | 100.00% | 2.60% | 96.70% |
SRR1232313_2.fastq.gz_trimming_report.txt | 32789834 | 56.70% | 100.00% | 2.60% | 96.60% |
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
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% |
Bsmap Alignment
- Bsmap can use 16 CPU, however, with only 8G memory. it is different with bismark. in bismark memory usage is proportional with CUP number with 3G.
#!/bin/csh #PBS -n Xie.LUN.SRR1232302_1.fastq.gz #PBS -q glean #PBS -l nodes=1:ppn=16 #PBS -l walltime=72:00:00 #PBS -o SRR1232302_1.fastq.gz.trim.log #PBS -e SRR1232302_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 bsmap -u -s 12 -v 0.04 -p 16 -a ../fastq2_trim/SRR1232302_1_val_1.fq.gz -b ../fastq2_trim/SRR1232302_2_val_2.fq.gz -d /home/shg047/db/hg19/meth/bismark/hg19.fa -o ../bsmap/SRR1232302_1.bam
- two job failed since glean. and this time pdafm were used to re-alignment. Resubmit (Feb/18/2016)
cd /home/shg047/oasis/Xliu2014/fastq qsub SRR1232310_1.fastq.gz.job qsub SRR1232312_1.fastq.gz.job
- two job completed (Feb/16/2016) and all the alignment were succeeded.the updated mapping statisitic
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 | 10879469 | 8166399 | 75.10% | 283705221 | 14059544 | 396832 | 1083414 | 14827795 | 65987979 | 187349657 | 48.70% | 0.60% | 0.60% |
SRR1232303_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 20185783 | 15643793 | 77.50% | 538753835 | 24467542 | 731361 | 1999839 | 29198598 | 125132925 | 357223570 | 45.60% | 0.60% | 0.60% |
SRR1232304_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 11064669 | 8668123 | 78.30% | 301062107 | 14716791 | 435437 | 1166303 | 16357108 | 70648275 | 197738193 | 47.40% | 0.60% | 0.60% |
SRR1232305_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 19379802 | 11358953 | 58.60% | 405848758 | 21963190 | 606473 | 1550397 | 22103090 | 96959575 | 262666033 | 49.80% | 0.60% | 0.60% |
SRR1232306_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 51331076 | 39232137 | 76.40% | 1375770206 | 70954836 | 1959550 | 5129368 | 73767418 | 324505783 | 899453251 | 49.00% | 0.60% | 0.60% |
SRR1232307_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 14987691 | 8977542 | 59.90% | 312378988 | 14249586 | 467917 | 1334605 | 15164065 | 70650657 | 210512158 | 48.40% | 0.70% | 0.60% |
SRR1232308_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 35378719 | 20706958 | 58.50% | 723041259 | 35238860 | 1069487 | 2948419 | 35619931 | 165087550 | 483077012 | 49.70% | 0.60% | 0.60% |
SRR1232309_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 34228833 | 20426178 | 59.70% | 704014483 | 32162156 | 1007755 | 2905755 | 30199793 | 156286525 | 481452499 | 51.60% | 0.60% | 0.60% |
SRR1232310_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 23850149 | 17995294 | 75.50% | 563331726 | 27402528 | 831180 | 2213192 | 31796570 | 132428948 | 368659308 | 46.30% | 0.60% | 0.60% |
SRR1232311_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 12142320 | 8808806 | 72.50% | 272573717 | 13022456 | 405024 | 1118769 | 14270047 | 63239639 | 180517782 | 47.70% | 0.60% | 0.60% |
SRR1232312_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 6133332 | 4094748 | 66.80% | 142984922 | 6981841 | 202170 | 531307 | 8206242 | 33563875 | 93499487 | 46.00% | 0.60% | 0.60% |
SRR1232313_1_val_1.fq.gz_bismark_bt2_PE_report.txt | 32727127 | 23917117 | 73.10% | 833607030 | 42703068 | 1162364 | 3069766 | 42946919 | 194253467 | 549471446 | 49.90% | 0.60% | 0.60% |
unSortBam to Methylfreq (Pair-end)
When you save pair-end reads into one bam file, usually the read1 and read2 would be one by one in the order. However, when you use samtools sort, this order will be changed and therefore, some further analysis would be report error when they require pair-wise order reads, such as bismark_methylation_extractor
#!/bin/csh #PBS -N SRR1232311_1_val_1.fq.gz_bismark_bt2_pe.bam #PBS -q glean #PBS -l nodes=1:ppn=6 #PBS -l walltime=6:00:00 #PBS -o SRR1232311_1_val_1.fq.gz_bismark_bt2_pe.bam.log #PBS -e SRR1232311_1_val_1.fq.gz_bismark_bt2_pe.bam.err #PBS -V #PBS -M shihcheng.guo@gmail.com #PBS -m abe #PBS -A k4zhang-group cd /oasis/tscc/scratch/shg047/Xliu2014/bam bismark_methylation_extractor --paired-end --bedGraph --multicore 3 --ignore 3 --ignore_3prime 3 --ignore_r2 5 --ignore_3prime_r2 5 --gzip --buffer_size 4G --zero_based --comprehensive --output ../methyfreq SRR1232311_1_val_1.fq.gz_bismark_bt2_pe.bamrm CHG* CpG* CHH* *bedGraph.gz *bismark.cov.gz
Detail Parameters as Following:
Parameters used to extract methylation information: Bismark Extractor Version: v0.14.4 Bismark result file: paired-end (SAM format) Ignoring first 3 bp of Read 1 Ignoring first 5 bp of Read 2 Ignoring last 3 bp of Read 1 Ignoring last 5 bp of Read 2 Output specified: comprehensive No overlapping methylation calls specified
Collect 0-based Coverage File:
Bam to SortBam (Feb 18 2016 Finished)
/home/shg047/oasis/Xliu2014/sortbam/SRR1232310.sort.bam
- two bam file was not intact and therefore alignment were re-done to SRR1232310_1 and SRR1232312_1