Under Construction! (Spring 2016)
Either visit our download page or clone our git repo.
$ git clone https://github.com/robinmeyers/transloc_pipeline.gitAdd directories to $PATH in ~/.profile or ~/.bash_profile
$ echo 'export PATH=~/transloc_pipeline/bin:~/transloc_pipeline/R:$PATH' >> ~/.bash_profileThe pipeline requires both a fasta file and a bowtie2 index of your reference genome. The pipeline will search for these elements in the locations specified by two environment variables: $GENOME_DB and $BOWTIE2_INDEXES. The pipeline will throw an error if it cannot find the bowtie2 index files at $BOWTIE2_INDEXES/<reference id> and the genome fasta at $GENOME_DB/<reference id>/<reference id>.fa.
First decide on your paths (e.g. ~/genomes or /usr/local/genomes for $GENOME_DB) and then add required environment variables to in ~/.profile or ~/.bash_profile
$ echo 'export GENOME_DB=~/genomes' >> ~/.bash_profile
$ echo 'export BOWTIE2_INDEXES=~/genomes/bowtie2_indexes' >> ~/.bash_profile
$ source ~/.bash_profileThe code below is one way to set up the pipeline to run against the hg19 reference genome. Running this will take some time to download the bowtie2 index and write the fasta file.
$ mkdir -p $BOWTIE2_INDEXES
$ cd $BOWTIE2_INDEXES
$ wget ftp://ftp.ccb.jhu.edu/pub/data/bowtie2_indexes/hg19.zip
$ unzip hg19.zip
$ mkdir -p $GENOME_DB/hg19
$ bowtie2-inspect hg19 > $GENOME_DB/hg19/hg19.fa There are lots. Sorry about that.
Install Bowtie2
If on OS X and using homebrew:
$ brew tap homebrew/science
$ brew install bowtie2Install Samtools
IMPT: The perl module that uses the samtools installation requires an older version of samtools. From the (README)[http://cpansearch.perl.org/src/LDS/Bio-SamTools-1.43/README], "This is a Perl interface to the SAMtools sequence alignment interface. It ONLY works on versions of Samtools up to 0.1.17. It does not work on version 1.0 or higher due to major changes in the library structure."
On OS X, I have had good success with the ActivePerl distribution. Perlbrew is a good option for a Linux machine, especially without root access.
Install Bioperl
Open an interactive cpan shell:
$ cpanFind the most recent version of bioperl:
cpan> d /bioperl/
Distribution CJFIELDS/BioPerl-1.6.901.tar.gz
Distribution CJFIELDS/BioPerl-1.6.923.tar.gz
Distribution CJFIELDS/BioPerl-1.6.924.tar.gzNow install:
cpan> install CJFIELDS/BioPerl-1.6.924.tar.gzor
cpan> force install CJFIELDS/BioPerl-1.6.924.tar.gzOther modules (all of which can be installed using cpan install commands)
cpan > install Getopt::Long Text::CSV List::MoreUtils Data::GUID Interpolation IPC::System::Simple Storable Switch::PlainI have missed a few. Missing modules can be installed the same as above.
SeqPrep is used in the pre-processing of the libraries.
$ git clone https://github.com/jstjohn/SeqPrep.git
$ cd SeqPrep
$ makeThen add SeqPrep to your $PATH.
Download and install R
Load an R session:
$ RInstall required packages:
> install.packages(c("magrittr", "readr", "stringr", "plyr", "dplyr", "data.table", ))
> source("https://bioconductor.org/biocLite.R")
> biocLite(c("GenomicRanges", "BSGenome"))or
$ git clone -b tutorial-data https://github.com/robinmeyers/transloc_pipeline.git tutorial_data
$ cd ./tutorial_dataThe Alt Lab primarily uses in-line barcodes (sequenced by the MiSeq at the head of the forward read) and then deconvolutes pooled libraries using this program. This script also calls an external tool to trim Illumina adapter sequence. If using Illumina multi-plex barcoding strategy, this script will not be useful except for trimming adapters, which is still recommended.
Deconvolutes and trims adapters
$ TranslocPreprocess.pl tutorial_metadata.txt preprocess/ --read1 pooled_R1.fq.gz --read2 pooled_R2.fq.gzJust trims adapters
$ cd ~/transloc_pipeline/data
$ TranslocPreprocess.pl tutorial_metadata.txt preprocess/ --indir ./$ TranslocWrapper.pl tutorial_metadata.txt preprocess/ results/ --threads 2The TranslocPipeline main output is the *.tlx file. One master tlx file will be generated per library. It can be thought of as similar to a sam file for NGS libraries. It contains output for every read in the library, and will serve as the starting point for all down-stream analyses. However, for most analyses, it will need to be filtered into only the required reads for the specific analysis. There is a default filtering that the pipeline will generate automatically. For any other filtering regime, the user will have to re-filter the master tlx file.
- unaligned: No OCS alignments.
- baitonly: Bait alignment is either the only alignent in the OCS or only followed by adapter alignment.
- uncut: Bait alignment runs greater than some number of bases past the cutsite.
- misprimed: Bait alignment runs fewer than some number of bases past the primer.
- freqcut: Restriction enzyme site within some number of bases from the junction. (Fairly depricated.)
- largegap: More than some number of bases between the bait and prey alignments.
- mapqual: OCS had a competing prey junction.
- breaksite: Prey alignment maps into non-endogenous breaksite cassette. (Fairly depricated.)
- sequential: Junction occurs downstream on read from first bait-prey junction.
$ TranslocFilter.pl results/RF204_Alt055/RF204_Alt055.tlx results/RF204_Alt055_refiltered.tlx --filters ""$ TranslocFilter.pl results/RF204_Alt055/RF204_Alt055.tlx results/RF204_Alt055_refiltered.tlx --filters ""$ TranslocFilter.pl results/RF204_Alt055/RF204_Alt055.tlx results/RF204_Alt055_refiltered.tlx --filters ""Two methods of detecting hotspots.
Must have MACS2 installed.
$ tlx2BED-MACS.pl
$ macs2$ TranslocHotspots.RAnd other viz.