Ns126:Xliu2014GB

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Revision as of 19:14, 20 January 2016 by >Shicheng (→‎Sample)
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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


Method

Sample

  • GEO Sample Records
title geo_accession channel_count tissue patient id smoking Gender Alignment processing sequencing library relation relation
75A-2 GSM1367123 1 adjacent nomal lung tissue 75 smoker male bsmap MethylPurify HiSeq 2000 Bisulfite-Seq SAMN02725476 SRX515122
75B GSM1367124 1 lung adenocarcinoma tissue 75 smoker male bsmap MethylPurify HiSeq 2001 Bisulfite-Seq SAMN02725471 SRX515123
109A-2 GSM1367125 1 adjacent nomal lung tissue 109 non-smoker male bsmap MethylPurify HiSeq 2002 Bisulfite-Seq SAMN02725468 SRX515124
109B GSM1367126 1 lung adenocarcinoma tissue 109 non-smoker male bsmap MethylPurify HiSeq 2003 Bisulfite-Seq SAMN02725475 SRX515125
109C GSM1367127 1 corresponding lymph node metastasis tissue 109 non-smoker male bsmap MethylPurify HiSeq 2004 Bisulfite-Seq SAMN02725470 SRX515126
137A GSM1367128 1 adjacent nomal lung tissue 137 non-smoker female bsmap MethylPurify HiSeq 2005 Bisulfite-Seq SAMN02725467 SRX515127
137B GSM1367129 1 lung adenocarcinoma tissue 137 non-smoker female bsmap MethylPurify HiSeq 2006 Bisulfite-Seq SAMN02725477 SRX515128
137C GSM1367130 1 corresponding lymph node metastasis tissue 137 non-smoker female bsmap MethylPurify HiSeq 2007 Bisulfite-Seq SAMN02725469 SRX515129
156A-2 GSM1367131 1 adjacent nomal lung tissue 156 non-smoker female bsmap MethylPurify HiSeq 2008 Bisulfite-Seq SAMN02725472 SRX515130
156B-2 GSM1367132 1 lung adenocarcinoma tissue 156 non-smoker female bsmap MethylPurify HiSeq 2009 Bisulfite-Seq SAMN02725478 SRX515131
201A GSM1367133 1 adjacent nomal lung tissue 201 non-smoker female bsmap MethylPurify HiSeq 2010 Bisulfite-Seq SAMN02725474 SRX515132
201B GSM1367134 1 lung adenocarcinoma tissue 201 non-smoker female bsmap MethylPurify HiSeq 2011 Bisulfite-Seq SAMN02725473 SRX515133
  • SRA Sample Records
Organism Sample File Name Size Updated
Homo sapiens SAMN02725476 SRR1232302 88.0Mb 2015/12/28 13:46
Homo sapiens SAMN02725471 SRR1232303 121.2Mb 2015/12/28 13:46
Homo sapiens SAMN02725468 SRR1232304 94.0Mb 2015/12/28 13:41
Homo sapiens SAMN02725475 SRR1232305 110.6Mb 2015/12/28 13:42
Homo sapiens SAMN02725470 SRR1232306 214.8Mb 2015/12/28 13:47
Homo sapiens SAMN02725467 SRR1232307 100.5Mb 2015/12/28 13:36
Homo sapiens SAMN02725477 SRR1232308 168.1Mb 2015/12/28 13:39
Homo sapiens SAMN02725469 SRR1232309 188.0Mb 2015/12/28 13:42
Homo sapiens SAMN02725472 SRR1232310 113.0Mb 2015/12/28 13:44
Homo sapiens SAMN02725478 SRR1232311 72.3Mb 2015/12/28 13:35
Homo sapiens SAMN02725474 SRR1232312 74.3Mb 2015/12/22 23:57
Homo sapiens SAMN02725473 SRR1232313 151.7Mb 2015/12/28 13:35

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 -q glean
#PBS -l nodes=1:ppn=16
#PBS -l walltime=72:00:00
#PBS -o SRR1232304_2.log
#PBS -e SRR1232304_2.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 --phred33-quals --fastq -L 20 -N 1 --multicore 6 /home/shg047/db/hg19/meth/bismark ../fastq_trim/SRR1232304_2_trimmed.fq.gz -o ../bam

Bam to Methylfreq