General installation for CBW tutorial

Environment setup

For this tutorial, we now assume the environment variable $TUTORIAL_HOME has been set to an existing directory to which the user has write access.

All binaries used in this tutorial will be installed using conda. Modify the PATH environment variable to point to binaries in the anaconda installation.

export PATH="/home/ubuntu/CourseData/CG_data/Module4/anaconda/bin:$PATH"

Installation

Tutorial directory structure

Create a directory for the reference data.

mkdir -p $TUTORIAL_HOME/refdata

Install tutorial scripts

All content for this tutorial is located in a bitbucket repo at https://dranew@bitbucket.org/dranew/cbw_tutorial.git. Some of the data, scripts and config files from this repo will be used in the tutorial. Clone the tutorial repo so we have a copy of the tutorial scripts in a known location.

cd $TUTORIAL_HOME/

git clone https://bitbucket.org/dranew/cbw_tutorial.git

Install Anaconda

All binaries used in this tutorial will be installed using conda. Download and install anaconda with the prefix $TUTORIAL_HOME/anaconda.

Packages in conda are stored in channels. Several additional channels hosting bioinformatics specific software will be required. Add additional channels using conda config.

conda config --add channels r
conda config --add channels bioconda
conda config --add channels BioBuilds
conda config --add channels https://conda.anaconda.org/dranew

Install samtools

The samtools package is the most widely used software for manipulating high-throughput sequence data stored in the ‘bam’ format.

Install samtools using conda.

conda install samtools

Install picard tools

Picard tools is a useful set of utilities for manipulating sequence data in bam/sam format.

Install picard using conda.

conda install picard

Install igv tools

The igvtools package provides utilities for preprocessing bam files for quicker viewing in IGV.

Install igvtools using conda.

conda install igvtools

Install bowtie and bowtie2

Install bowtie and bowtie2 using conda.

conda install bowtie bowtie2

Install the gmap aligner

Install gmap using conda.

conda install gmap

Install the bwa aligner

Install bwa using conda.

conda install bwa

Installation of the ChimeraScan gene fusion prediction tool

Environment setup

Set variable for index directory.

CHIMERASCAN_INDEX=$TUTORIAL_HOME/refdata/chimerascan/indices/

Installation

Install ChimeraScan

Install in ChimeraScan using conda.

conda install chimerascan

Install the required reference data files

Install the reference data in a subdirectory of the tutorial ref data.

mkdir -p $TUTORIAL_HOME/refdata/chimerascan/
cd $TUTORIAL_HOME/refdata/chimerascan/

Download the gene models from chimerascan’s google code site as specified in the instructions.

wget https://chimerascan.googlecode.com/files/hg19.ucsc_genes.txt.gz
gunzip hg19.ucsc_genes.txt.gz

Build the chimerascan indices using the chimerascan_index.py command.

mkdir -p $CHIMERASCAN_INDEX
chimerascan_index.py \
    $UCSC_GENOME_FILENAME hg19.ucsc_genes.txt \
    $CHIMERASCAN_INDEX

Installation of the deFuse gene fusion prediction tool

Environment setup

Set variable for the config filename and the two scripts.

DEFUSE_CONFIG=$TUTORIAL_HOME/cbw_tutorial/config/defuse_chr1.txt
DEFUSE_REF_DATA=$TUTORIAL_HOME/refdata/defuse/

Installation

Install deFuse

Install in ChimeraScan using conda.

conda install defuse

Install the required reference data files

The reference data files can be downloaded and index automatically using the defuse_create_ref.pl script.

defuse_create_ref.pl -c $DEFUSE_CONFIG -d $DEFUSE_REF_DATA

Installation of the STAR RNA-Seq aligner

Environment

Specify the directory in which the reference genome data will be stored.

STAR_GENOME_INDEX=$TUTORIAL_HOME/refdata/star/
STAR_FUSION_GENOME_INDEX=$TUTORIAL_HOME/refdata/star/GRCh37_gencode_v19_CTAT_lib/

Installation

Install STAR using conda. Also install perl and the perl package Set::IntervalTree, required by STAR-Fusion.

conda install perl-threaded
conda install perl-set-intervaltree
conda install star

Create genome

For STAR-Fusion, we require an additional reference dataset.

cd $STAR_GENOME_INDEX

wget https://data.broadinstitute.org/Trinity/CTAT_RESOURCE_LIB/GRCh37_gencode_v19_CTAT_lib.tar.gz

tar -xvf GRCh37_gencode_v19_CTAT_lib.tar.gz

cd GRCh37_gencode_v19_CTAT_lib/

Optionally subset the reference for the tutorial chromosomes.

samtools faidx ref_genome.fa chr$TUTORIAL_CHROMOSOME \
    > ref_genome.chr$TUTORIAL_CHROMOSOME.fa
mv ref_genome.chr$TUTORIAL_CHROMOSOME.fa ref_genome.fa
rm ref_genome.fa.fai

grep -P "^chr$TUTORIAL_CHROMOSOME\t" ref_annot.gtf \
    > ref_annot.chr$TUTORIAL_CHROMOSOME.gtf
mv ref_annot.chr$TUTORIAL_CHROMOSOME.gtf ref_annot.gtf

Prepare the genome and annotations for star fusion.

prep_genome_lib.pl \
    --genome_fa ref_genome.fa \
    --gtf ref_annot.gtf \
    --blast_pairs blast_pairs.outfmt6.gz

Installation of the tophat-fusion gene fusion prediction tool

Environment setup

Location of tophat-fusion specific gene models, ensembl but with chr prefix.

TOPHAT_GTF_FILENAME=$TUTORIAL_HOME/refdata/tophatfusion/Homo_sapiens.GRCh37.75.gtf

Installation

Install tophat and tophat-fusion using conda:

conda install tophat

Reference Genome Preparation

Install the reference gene annotations provided by tophat-fusion in the tophat-fusion specific reference data directory.

mkdir -p $TUTORIAL_HOME/refdata/tophatfusion/
cd $TUTORIAL_HOME/refdata/tophatfusion/
wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz
gunzip refGene.txt.gz
wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/ensGene.txt.gz
gunzip ensGene.txt.gz

We require the gene models in GTF format and we will use the GTF provided by ensembl. However, we first need to add the ‘chr’ prefix to the chromosome names so we match the ucsc genome. Use the sed command to create a modified version of the ensembl gene models with the chr prefix for each chromosome. This can be done by just adding chr to the beginning of each line in the GTF file.

sed 's/^\([^#]\)/chr'$TUTORIAL_CHROMOSOME'/' $ENSEMBL_GTF_FILENAME | sed 's/^chrMT/chrM/' > $TOPHAT_GTF_FILENAME

Tophat requires additional blast databases, download and install these.

mkdir -p $TUTORIAL_HOME/refdata/tophatfusion/blast
cd $TUTORIAL_HOME/refdata/tophatfusion/blast
wget ftp://ftp.ncbi.nlm.nih.gov/blast/db/human_genomic.*.tar.gz
wget ftp://ftp.ncbi.nlm.nih.gov/blast/db/nt.*.tar.gz
gunzip *.gz

Installation of the Trinity RNA-Seq assembler

Installation

Install trinity using conda

conda install trinity