Author: Khalid Mahmood (kmahmood@unimelb.edu.au)

hiplexpipe is based on the Ruffus library for writing bioinformatics pipelines. Its features include:

• Job submission on a cluster using DRMAA (currently only tested with SLURM).
• Job dependency calculation and checkpointing.
• Pipeline can be displayed as a flowchart.
• Re-running a pipeline will start from the most up-to-date stage. It will not redo previously completed tasks.

See LICENSE.txt in source repository.

hiplexpipe depends on the following programs and libraries:

• python (version 2.7.5)
• java (version 1.8)
• DRMAA for submitting jobs to the cluster (it uses the Python wrapper to do this). You need to install your own libdrama.so for your local job submission system. There are versions available for common schedulers such as Torque/PBS, SLURM and so on.

• SAMtools (version 1.3.1)
• bwa for aligning reads to the reference genome (version 0.7.15)
• GATK for calling variants and genotyping (version 3.6)
• BEDTools for calculating sequencing coverage statistics (version 2.26.0)

hiplexpipe assumes the tools above are installed by the users themselves.

hiplexpipe depends on the following python libraries, tools and wrappers.

• Python 2.7.5
• PyVCF
• Biopython
• pybedtools
• cyvcf2

We recommend using a python virtual environment. Following is an examples of how to setup a hiplexpipe virtual environment ready for analysis:

module load Python/2.7.10-vlsci_intel-2015.08.25
export DRMAA_LIBRARY_PATH=/usr/local/slurm_drmaa/1.0.7-GCC/lib/libdrmaa.so
virtualenv --system-site-packages venv
source venv/bin/activate
pip install -U https://github.com/khalidm/undr_rover/archive/master.zip
pip install -U https://github.com/khalidm/hiplexpipe/archive/master.zip
hiplexpipe --config pipeline.config --use_threads --log_file pipeline.log --jobs 10 --verbose 3 --just_print

You should have two target interval files for every Hi-Plex experiment.

1. rover.txt - this contains the amplicon regions and primer sequences.
2. idt.txt - this file contains the primer sequences and their names matching the names in the above rover.txt file.

Follow instructions below to prepare the intervals files for the pipeline. (We are working on a tool to automate this task).

1. Main rover bed file. (rover.bed) This file is used to calculate alignment and coverage statistics. cut -f1,2,3,4,5 rover.txt > rover.bed or awk ' BEGIN{FS="\t";OFS="\t"}; { print $1,int($2+($3-$2)/2),int($3-($3-$2)/2),$4,$5} ' rover.txt > rover.bed
2. !Interval file. (rover.interval_list) - not required as the input bam is now clipped. java -jar picard.jar BedToIntervalList I=rover.bed SD=<hg19.dict> -O=rover.interval_list
3. Primer coordinates file. (primer.bedpe) This file is used to clip primer sequences from the alignments. awk ' BEGIN{FS="\t";OFS="\t"}; { print $1,$7,$8,$1,$12,$11} ' rover.txt > primer.bedpe