Ns126:Xliu2014GB

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Revision as of 19:07, 20 January 2016 by >Shicheng (→‎Method)
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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

title geo_accession type channel_count source_name_ch1 organism_ch1 characteristics_ch1 characteristics_ch1.1 characteristics_ch1.3 Alignment processing instrument_model library_strategy relation relation.1
75A-2 GSM1367123 SRA 1 adjacent nomal lung tissue Homo sapiens patient id: 75 smoker male bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725476 SRX515122
75B GSM1367124 SRA 1 lung adenocarcinoma tissue Homo sapiens patient id: 75 smoker male bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725471 SRX515123
109A-2 GSM1367125 SRA 1 adjacent nomal lung tissue Homo sapiens patient id: 109 non-smoker male bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725468 SRX515124
109B GSM1367126 SRA 1 lung adenocarcinoma tissue Homo sapiens patient id: 109 non-smoker male bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725475 SRX515125
109C GSM1367127 SRA 1 corresponding lymph node metastasis tissue Homo sapiens patient id: 109 non-smoker male bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725470 SRX515126
137A GSM1367128 SRA 1 adjacent nomal lung tissue Homo sapiens patient id: 137 non-smoker female bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725467 SRX515127
137B GSM1367129 SRA 1 lung adenocarcinoma tissue Homo sapiens patient id: 137 non-smoker female bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725477 SRX515128
137C GSM1367130 SRA 1 corresponding lymph node metastasis tissue Homo sapiens patient id: 137 non-smoker female bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725469 SRX515129
156A-2 GSM1367131 SRA 1 adjacent nomal lung tissue Homo sapiens patient id: 156 non-smoker female bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725472 SRX515130
156B-2 GSM1367132 SRA 1 lung adenocarcinoma tissue Homo sapiens patient id: 156 non-smoker female bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725478 SRX515131
201A GSM1367133 SRA 1 adjacent nomal lung tissue Homo sapiens patient id: 201 non-smoker female bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725474 SRX515132
201B GSM1367134 SRA 1 lung adenocarcinoma tissue Homo sapiens patient id: 201 non-smoker female bsmap MethylPurify Illumina HiSeq 2000 Bisulfite-Seq SAMN02725473 SRX515133

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