mate-pair QC
Version 0.1
2015-04-24

This pipeline is made available with no waranty of usefulness of any kind. It has been put together for quality control of mate-pair library conformation and insert sizes for use in scaffolding assemblies. This pipeline uses valuable tools developed by other groups (see 'Requires' below)

Quality trim mate pairs, bin by conformation MP/PE/Unkn, align to reference then estimate insert size

1. Trim for quality
2. Separate mate-pairs based on adapter presence and position
3. Index reference with bwa
4. Align mate-pairs against reference
5. Evaluate your mate-pair libraries

Requires the following:
Trimmomatic http://www.usadellab.org/cms/?page=trimmomatic
NxTrim https://github.com/sequencing/NxTrim
bwa http://bio-bwa.sourceforge.net
samtools http://samtools.sourceforge.net
picard tools http://broadinstitute.github.io/picard/

Raw *fastq.gz mate-pair data in 02_raw_data; run all jobs from the main directory. Job files are specific to Katak at IBIS, but with some minor editing can be adapted for other servers.

Generates a 'paired' and 'single' fastq file for each mate-pair library
requires Trimmomatic

Edit 01_scripts/01_trimming.sh by giving the path to trimmomatic
Run locally:

01_scripts/01_trimming.sh

Run on Katak:

qsub 01_scripts/jobs/01_trimming_job.sh

Bin quality trimmed paired mate-pairs for presence of adapter
requires NxTrim

Mate pairs can be in mate-pair conformation, paired-end conformation (i.e. shadow library) or unknown. NxTrim sorts each mate pair into these options based on presence of connecting adapter in forward or reverse read.

Edit 01_scripts/02_NxTrim_binning.sh by giving path to nxtrim

Locally:

01_scripts/02_NxTrim_binning.sh

On Katak:

qsub 01_scripts/jobs/02_NxTrim_binning_job.sh

Normally mate-pairs are in RF directionality, but NxTrim will revcomp and output as FR (ready for alignment)
The output of this will give *.mp.fastq.gz in 04_binned_mps

Note: only need to do this once
requires bwa

Locally: ensure reference is in fasta format (not compressed), and change REFERENCE to the path to your reference you want to align against

bwa index REFERENCE

On Katak:

qsub 01_scripts/jobs/03a_indexRef_job.sh

requires bwa and samtools

Edit 01_scripts/03b_BWAaln.sh by giving path and names to each sample and RG indexes (e.g. replace short identifier (bolded for clarity)) for each RG at RG[1]='@RG\tID:lib208\tSM:lib208\tPL:Illumina'

Locally:

01_scripts/03b_BWAaln.sh

On Katak:

qsub 01_scripts/jobs/03b_BWAaln_job.sh

samtools flagstat <your.bam>

requires picard tools locally:

./01_scripts/04_estInsertSizes.sh

On Katak:

qsub 01_scripts/jobs/04_estInsertSizes_job.sh

Enjoy! Hopefully your insert sizes are as you expect.