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bwa-parclip

bwaparclip is an experimental a hack based on bwameth to align PAR-CLIP reads without intermediate temp files. This works for single-end reads and for paired-end reads from the directional protocol. All praise should go Brent Pedersen for making bwa-meth, and all blame to me for mistakes introduced in bwa-parclip.

Uses the method of in silico conversion of all T's to C's in both reference and reads.

Recovers the original read by attaching it as a comment which bwa appends as a tag to the read.

QuickStart

Without installation, you can use as python bwaparclip.py with install, the command is bwaparclip.py.

The commands:

bwaparclip.py index $REF
bwaparclip.py --reference $REF some_R1.fastq.gz some_R2.fastq.gz --prefix some.output

will create some.output.bam and some.output.bam.bai. To align single end-reads, specify only 1 file.

Installation

The following snippet should work for most systems that have samtools and bwa installed and the ability to install python packages. (Or, you can send this to your sys-admin). See the dependencies section below for further instructions:

    # these 4 lines are only needed if you don't have toolshed installed
    wget https://pypi.python.org/packages/source/t/toolshed/toolshed-0.4.0.tar.gz
    tar xzvf toolshed-0.4.0.tar.gz
    cd toolshed-0.4.0
    sudo python setup.py install

    wget https://github.com/PeteHaitch/bwa-parclip/archive/v0.0.9000.tar.gz
    tar xzvf v0.0.9000.tar.gz
    cd bwa-parclip-0.0.9000/
    sudo python setup.py install

After this, you should be able to run: bwaparclip.py and see the help.

Dependencies

bwa-parclip depends on

  • python 2.7+ (including python3)

    • toolshed library. can be installed with:

      • easy_install toolshed or
      • pip install toolshed

    • if you don't have root or sudo priveleges, you can run python setup.py install --user from this directory and the bwaparclip.py executable will be at: ~/.local/bin/bwaparclip.py

    • if you do have root or sudo run: [sudo] python setup.py install from this directory

    • users unaccustomed to installing their own python packages should download anaconda: https://store.continuum.io/cshop/anaconda/ and then install the toolshed module with pip as described above.

  • samtools command on the $PATH (https://github.com/samtools/samtools)

  • bwa mem from: https://github.com/lh3/bwa

usage

Index

One time only, you need to index a reference sequence.

bwaparclip.py index $REFERENCE

If your reference is some.fasta, this will create some.c2t.fasta and all of the bwa indexes associated with it.

Align

bwaparclip.py --threads 16 \
     --prefix $PREFIX \
     --reference $REFERENCE \
     $FQ1 $FQ2

This will create $PREFIX.bam and $PREFIX.bam.bai. The output will pass Picard-tools ValidateSam and will have the reads in the correct location (flipped from G => A reference).

Handles clipped alignments and indels correctly. Fastqs can be gzipped or not.

The command above will be sent to BWA to do the work as something like:

bwa mem -L 25 -pCM -t 15  $REFERENCE.c2t.fa \
        '<python bwaparclip.py c2t $FQ1 $FQ2'

So the converted reads are streamed directly to bwa and never written to disk. The output from that is modified by bwa-parclip and streamed straight to a bam file.