The analysis scripts made available here were used in a study of the temporal response of the microbiome in plasma to perturbations of the immune system and antiviral treatments.
Organ transplantation was chosen as the model system for this study. Plasma DNA was sequenced from samples collected from organ transplant patients post transplant.
Title: Temporal Response of the Human Virome to Immunosuppression and Antiviral Therapy
Authors: Iwijn De Vlaminck,1 Kiran K. Khush,2 Calvin Strehl, Bitika Kohli, Norma F. Neff,1 Jennifer Okamoto,1 Thomas M. Snyder,1 David Weill,3 Daniel Bernstein,4 Hannah A. Valantine,2, and Stephen R. Quake1,*
1 Depts. of Bioengineering and Applied Physics, Stanford University and the Howard Hughes Medical Institute, Stanford, CA 94305
2 Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305
3 Division of Pulmonary and Ctical Care Medicine, Stanford University School of Medicine, Stanford, CA 94305
4 Division of Pediatric Cardiology, Stanford University School of Medicine, Stanford, CA 94305
The work was supported by NIH grant RC4AI092673 and the Howard Hughes Medical Institute.
Many of the scripts made available here are compatible with the Sun Grid Egine bacth-queuing system (http://en.wikipedia.org/wiki/Oracle_Grid_Engine) and can be run on a parallel computing cluster.
Where available we provide the scripts in a SGE-compatible format.
Folder FilterHost:
Microbiome-derived sequences are identified after computational subtraction of human-derived sequences (Weber et al., 2002; Xu et al., 2003).
Duplicate and low quality reads are removed and the remaining reads are mapped to the human reference genome, build hg19 (BWA (Li and Durbin, 2009).
Unmapped reads are collected and low complexity reads removed (SEQCLEAN, http://www.tigr.org/tdb/tgi/software).
> clean.sge.sh
Exact duplicates are removed using the C-based utility fastq.cpp. Low quality reads were removed using the quality filter that is part of the fastx package.
> aln.sge.sh and samse.sge.sh
The remaining reads are subsequently aligned using BWA to the human reference genome build hg19 (bwaaln - q25). see: aln.sge.sh and samse.sge.sh
> filter.sge.sh
Unmapped reads were collected using samtools (samtools view -f4) and low complexity reads were removed using Seqclean (seqclean -l 40 -c 1, see https://sourceforge.net/projects/seqclean/).
Folder SpeciesAbundance
Reads were subsequently aligned using BLAST to a selection of viral, baterial and fungal reference genomes (ncbi_viruses/all.fna.tar.gz, ncbi_bacteria/all.fna.tar.gz and all references in ncbi_fungi downloaded from ftp://ftp.ncbi.nih.gov/genomes on June 14, 2012).
> blast.sge.sh and blast.sh
Relative genome abundance estimation was calculated with GRAMMy (Xia et al. 2011). This tool utilizes the BLAST-derived nucleic acid sequence-similarity data to perform a maximum likelihood estimation of the relative abundance of species in the sample. GRAMMy filters hits by BLAST alignment metrics (E-score, alignment length and identity rate) and accounts for the target genome size and the ambiguity of read assignments in assessing the relative abundance of the candidate reference genomes. > grammy.sge.sh
The grammy results files are re-organized.
> lineage.sge.sh
Abudance data for a list of taxonomic nodes are extracted
> taxonomy.sge.sh
The folder Taxonomy contains scripts for the compilation of a blast database and a reference (gdt file) for Grammy given a fasta file.
The folder furthermore contains a script for the extraction of a minimal taxonomy and structure of the phylogenetic tree given a blast database
