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WGS pipeline for microbiome profiling from biopsy samples

Computational pipelines for filtering host DNA and evaluating the microbiome abundance from biopsy WGS samples.

Citing our paper:

Zhang C, Cleveland K, Schnoll-Sussman F, McClure B, Bigg M, Thakkar P, Schultz M, Shah M, and Betel D. Identification of the gastric microbiome from endoscopic biopsy samples using whole genome sequencing. Genome Biology. 2015

Installing prerequisite software and corresponding databases

BWA

Download: BWA 0.6.2
hg19 Human reference genome: http://hgdownload.soe.ucsc.edu/goldenPath/hg19/chromosomes/
Additional human genomes: ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens//ARCHIVE/BUILD.37.3/Assembled_chromosomes/seq/

RepearMasker

Download: RepeatMasker 4.0.5
Database: [Repbase for RepeatMasker] (http://www.girinst.org/)

BLAST & MegaBLAST 2.2.27

Download: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.27/
Database: [Combined human sequence database] (http://www.girinst.org/)

Bowtie2

Download: Bowtie2 2.2.5
NCBI bacteria compelete genomes: ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/

PathoScope

Download: PathoScope 2.0

R

Download: R 3.0.2

samtools

Download: samtools 0.1.19

Setting the parameters

Set the correct paths to above software and databases in configs.sh, and run bash configs.sh

Filtering host DNA

Step 1. Align all reads to hg19 Human reference genome with BWA
bash Filter_S1.sh
Step 2. Align the remaining unmapped reads to additional human genomes with BWA
bash Filter_S2.sh
Step 3. Filter the remaining unmapped reads with RepearMasker
bash Filter_S3.sh
Step 4. Align the remaining reads to human sequence database with BLAST
bash Filter_S4.sh
Step 5. Align the remaining reads to human sequence database with MegaBLAST
bash Filter_S5.sh

Microbiome profiling

Step 1. Align fully filtered reads to NCBI bacteria compelete genomes with Bowtie2
Step 2. Calculate the genome coverages to further remove false identification
Step 3. Assign the multiple mapped the reads with PathoScope
Step 4. Re-evaluate the relative abundances of microbiome

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