Mapping reads with Mosaik - Revision history

Hjmegens: /* Align reads with MOSAIK */

2012-03-04T19:19:19Z

Align reads with MOSAIK

← Older revision		Revision as of 21:19, 4 March 2012
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	{\| width="100%"\|		{\| width="100%"\|
	<source lang='bash'>		<source lang='bash'>
	MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia $LIBPATH/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j $LIBPATH/Mosaiklibs/Ssc10_2_wUn.jump15		MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia $LIBPATH/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 \
			-p 24 -act 20 -j $LIBPATH/Mosaiklibs/Ssc10_2_wUn.jump15
	</source>		</source>
	\|}		\|}

	=== Sort the alignment file ===		=== Sort the alignment file ===
	<source lang='bash'>		<source lang='bash'>

Hjmegens at 15:43, 25 November 2011

2011-11-25T15:43:04Z

← Older revision		Revision as of 17:43, 25 November 2011
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	MosaikSort -in aligned_reads.dat -out aligned_reads-sorted.dat		MosaikSort -in aligned_reads.dat -out aligned_reads-sorted.dat
	</source>		</source>
	==== convert to BAM ===		=== convert to BAM ===
	<source lang='bash'>		<source lang='bash'>
	MosaikText -in aligned_reads-sorted.dat -bam aligned_reads-sorted.bam		MosaikText -in aligned_reads-sorted.dat -bam aligned_reads-sorted.bam

Hjmegens at 15:41, 25 November 2011

2011-11-25T15:41:38Z

← Older revision		Revision as of 17:41, 25 November 2011
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	=== Align reads with MOSAIK ===		=== Align reads with MOSAIK ===
	{\| width="100%"		{\| width="100%"\|
	~~\|- valign="top"~~
	~~\| width="25~~%" \|
	<source lang='bash'>		<source lang='bash'>
	MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia $LIBPATH/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j $LIBPATH/Mosaiklibs/Ssc10_2_wUn.jump15		MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia $LIBPATH/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j $LIBPATH/Mosaiklibs/Ssc10_2_wUn.jump15

Hjmegens at 15:40, 25 November 2011

2011-11-25T15:40:25Z

← Older revision		Revision as of 17:40, 25 November 2011
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	\|}		\|}
	=== Sort the alignment file ===		=== Sort the alignment file ===
	<~~pre~~>		<source lang='bash'>
	MosaikSort -in aligned_reads.dat -out aligned_reads-sorted.dat		MosaikSort -in aligned_reads.dat -out aligned_reads-sorted.dat
	</~~pre~~>		</source>
	==== convert to BAM ===		==== convert to BAM ===
	<~~pre~~>		<source lang='bash'>
	MosaikText -in aligned_reads-sorted.dat -bam aligned_reads-sorted.bam		MosaikText -in aligned_reads-sorted.dat -bam aligned_reads-sorted.bam
	</~~pre~~>		</source>
	== Finalizing preparation of BAM files with SAMtools ==		== Finalizing preparation of BAM files with SAMtools ==
	=== prepare a new header file ===		=== prepare a new header file ===
	<~~pre~~>		<source lang='bash'>
	samtools view -H aligned_reads-sorted.bam \| sed 's/SM:unknown/SM:pigname/' \| sed 's/PL:sanger/PL:ILLUMINA/' >newheader.txt		samtools view -H aligned_reads-sorted.bam \| sed 's/SM:unknown/SM:pigname/' \| sed 's/PL:sanger/PL:ILLUMINA/' >newheader.txt
	</~~pre~~>		</source>

	<~~pre~~>		<source lang='bash'>
	samtools view -H another_aligned_reads-sorted.bam \| sed 's/SM:unknown/SM:pigname/' \| sed 's/PL:sanger/PL:ILLUMINA/' \| grep @RG >>newheader.txt		samtools view -H another_aligned_reads-sorted.bam \| sed 's/SM:unknown/SM:pigname/' \| sed 's/PL:sanger/PL:ILLUMINA/' \| grep @RG >>newheader.txt
	</~~pre~~>		</source>

	=== merge BAM files (if applicable) ===		=== merge BAM files (if applicable) ===
	<~~pre~~>		<source lang='bash'>
	samtools merge merged.bam aligned_reads-sorted.bam another_aligned_reads-sorted.bam		samtools merge merged.bam aligned_reads-sorted.bam another_aligned_reads-sorted.bam
	</~~pre~~>		</source>

	=== reheader merged BAM file ===		=== reheader merged BAM file ===
	<~~pre~~>		<source lang='bash'>
	samtools reheader newheader.txt merged.bam >pigname_rh.bam		samtools reheader newheader.txt merged.bam >pigname_rh.bam
	</~~pre~~>		</source>

	== Variant calling with Samtools ==		== Variant calling with Samtools ==
	=== calling raw variants ===		=== calling raw variants ===
	<~~pre~~>		<source lang='bash'>
	samtools view -u pigname_rh.bam \| samtools pileup -vcf /shared/Sus/Sscrofa_build10_2/FASTA/Ssc10_2_wUn.fa - >vars-raw_pigname.txt		samtools view -u pigname_rh.bam \| samtools pileup -vcf /shared/Sus/Sscrofa_build10_2/FASTA/Ssc10_2_wUn.fa - >vars-raw_pigname.txt
	</~~pre~~>		</source>
	=== filtering variants ===		=== filtering variants ===

	<~~pre~~>		<source lang='bash'>
	samtools.pl varFilter -D25 vars-raw_pigname.txt \| awk '($3==""&&$6>=50)\|\|($3!=""&&$6>=20)' >vars-flt_pigname-final.txt		samtools.pl varFilter -D25 vars-raw_pigname.txt \| awk '($3==""&&$6>=50)\|\|($3!=""&&$6>=20)' >vars-flt_pigname-final.txt
	</~~pre~~>		</source>

	== Additional notes ==		== Additional notes ==
	=== submitting using grid-engine ===		=== submitting using grid-engine ===
	Submitting jobs using the grid-engine (qsub) requires a few additional options. The following lines can be added to the script to be submitted:		Submitting jobs using the grid-engine (qsub) requires a few additional options. The following lines can be added to the script to be submitted:
	<~~pre~~>		<source lang='bash'>
	#!/bin/bash		#!/bin/bash
	#$ -cwd		#$ -cwd
	#$ -S /bin/sh		#$ -S /bin/sh
	#$ -v MOSAIK_TMP=/srv/nfs02/yourhomedir/		#$ -v MOSAIK_TMP=/srv/nfs02/yourhomedir/
	</~~pre~~>		</source>

	=== Tidying up ===		=== Tidying up ===
	Please clean up the intermediate files as soon as they are not needed any more.		Please clean up the intermediate files as soon as they are not needed any more.
	<~~pre~~>		<source lang='bash'>
	rm *.dat		rm *.dat
	rm reads.bam		rm reads.bam
	</~~pre~~>		</source>

Hjmegens at 15:38, 25 November 2011

2011-11-25T15:38:31Z

← Older revision		Revision as of 17:38, 25 November 2011
Line 9:		Line 9:
	=== reference assembly files for MOSAIK ===		=== reference assembly files for MOSAIK ===
	The procedure described here requires a number of MOSAIK specific input files: a '.dat' file of the reference assembly, and a so called 'jump' database. These can be found in:		The procedure described here requires a number of MOSAIK specific input files: a '.dat' file of the reference assembly, and a so called 'jump' database. These can be found in:
	<~~pre~~>		<source lang='bash'>
	/shared/Sus/Sscrofa_build10_2/		LIBPATH='/shared/Sus/Sscrofa_build10_2/'
	</~~pre~~>		</source>

	== Mapping procedure ==		== Mapping procedure ==
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	\| width="25%" \|		\| width="25%" \|
	<source lang='bash'>		<source lang='bash'>
	MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia ~~/shared/Sus/Sscrofa_build10_2~~/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j ~~/shared/Sus/Sscrofa_build10_2~~/Mosaiklibs/Ssc10_2_wUn.jump15		MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia $LIBPATH/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j $LIBPATH/Mosaiklibs/Ssc10_2_wUn.jump15
	</source>		</source>
	\|}		\|}

Hjmegens at 15:36, 25 November 2011

2011-11-25T15:36:13Z

← Older revision		Revision as of 17:36, 25 November 2011
Line 16:		Line 16:
	=== creating MOSAIK file for the reads ===		=== creating MOSAIK file for the reads ===
	The first step is creating '.dat' file of the sequence reads.		The first step is creating '.dat' file of the sequence reads.
	<~~pre~~>		<source lang='bash'>
	MosaikBuild -q reads_1_QT.fq.gz -q2 reads_2_QT.fq.gz -out reads_QT.dat -st sanger		MosaikBuild -q reads_1_QT.fq.gz -q2 reads_2_QT.fq.gz -out reads_QT.dat -st sanger
	</~~pre~~>		</source>

	=== Align reads with MOSAIK ===		=== Align reads with MOSAIK ===
Line 24:		Line 24:
	\|- valign="top"		\|- valign="top"
	\| width="25%" \|		\| width="25%" \|
	<~~pre~~>		<source lang='bash'>
	MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.jump15		MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.jump15
	</~~pre~~>		</source>
	\|}		\|}
	=== Sort the alignment file ===		=== Sort the alignment file ===

Hjmegens at 15:34, 25 November 2011

2011-11-25T15:34:21Z

← Older revision		Revision as of 17:34, 25 November 2011
Line 23:		Line 23:
	{\| width="100%"		{\| width="100%"
	\|- valign="top"		\|- valign="top"
			\| width="25%" \|
	<pre>		<pre>
	MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.jump15		MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.jump15
	</pre>		</pre>
	\|}		\|}
	==== Sort the alignment file ===		=== Sort the alignment file ===
	<pre>		<pre>
	MosaikSort -in aligned_reads.dat -out aligned_reads-sorted.dat		MosaikSort -in aligned_reads.dat -out aligned_reads-sorted.dat

Hjmegens at 15:33, 25 November 2011

2011-11-25T15:33:06Z

← Older revision		Revision as of 17:33, 25 November 2011
Line 21:		Line 21:

	=== Align reads with MOSAIK ===		=== Align reads with MOSAIK ===
			{\| width="100%"
			\|- valign="top"
	<pre>		<pre>
	MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.jump15		MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.jump15
	</pre>		</pre>
			\|}
	==== Sort the alignment file ===		==== Sort the alignment file ===
	<pre>		<pre>

Hjmegens: Created page with "Within the Pig ERC project we have several mapping strategies. This article describes the mapping procedure using Mosaik for paired-end mapping to the reference assembly. == Pre..."

2011-11-24T15:45:31Z

Created page with "Within the Pig ERC project we have several mapping strategies. This article describes the mapping procedure using Mosaik for paired-end mapping to the reference assembly. == Pre..."

New page

Within the Pig ERC project we have several mapping strategies. This article describes the mapping procedure using Mosaik for paired-end mapping to the reference assembly.

== Prerequisites ==
=== input reads ===
The mapping procedure requires paired end reads in fastq format. For the current procedure quality-trimmed reads are used. Forward and reverse reads should be in two separate files, and should be in the same order. Input files are assumed to have Sanger-style quality scaling.

Here the input fastq files are named 'reads_1_QT.fq.gz', for forward reads, and 'reads_2_QT.fq.gz' for reverse reads.

=== reference assembly files for MOSAIK ===
The procedure described here requires a number of MOSAIK specific input files: a '.dat' file of the reference assembly, and a so called 'jump' database. These can be found in:
<pre>
/shared/Sus/Sscrofa_build10_2/
</pre>

== Mapping procedure ==
=== creating MOSAIK file for the reads ===
The first step is creating '.dat' file of the sequence reads.
<pre>
MosaikBuild -q reads_1_QT.fq.gz -q2 reads_2_QT.fq.gz -out reads_QT.dat -st sanger
</pre>

=== Align reads with MOSAIK ===
<pre>
MosaikAligner -in reads_QT.dat -out aligned_reads.dat -ia /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.dat -hs 15 -mmp 0.07 -m all -mhp 10 -p 24 -act 20 -j /shared/Sus/Sscrofa_build10_2/Mosaiklibs/Ssc10_2_wUn.jump15
</pre>

==== Sort the alignment file ===
<pre>
MosaikSort -in aligned_reads.dat -out aligned_reads-sorted.dat
</pre>
==== convert to BAM ===
<pre>
MosaikText -in aligned_reads-sorted.dat -bam aligned_reads-sorted.bam
</pre>
== Finalizing preparation of BAM files with SAMtools ==
=== prepare a new header file ===
<pre>
samtools view -H aligned_reads-sorted.bam | sed 's/SM:unknown/SM:pigname/' | sed 's/PL:sanger/PL:ILLUMINA/' >newheader.txt
</pre>

<pre>
samtools view -H another_aligned_reads-sorted.bam | sed 's/SM:unknown/SM:pigname/' | sed 's/PL:sanger/PL:ILLUMINA/' | grep @RG >>newheader.txt
</pre>

=== merge BAM files (if applicable) ===
<pre>
samtools merge merged.bam aligned_reads-sorted.bam another_aligned_reads-sorted.bam
</pre>

=== reheader merged BAM file ===
<pre>
samtools reheader newheader.txt merged.bam >pigname_rh.bam
</pre>

== Variant calling with Samtools ==
=== calling raw variants ===
<pre>
samtools view -u pigname_rh.bam | samtools pileup -vcf /shared/Sus/Sscrofa_build10_2/FASTA/Ssc10_2_wUn.fa - >vars-raw_pigname.txt
</pre>
=== filtering variants ===

<pre>
samtools.pl varFilter -D25 vars-raw_pigname.txt | awk '($3=="*"&&$6>=50)||($3!="*"&&$6>=20)' >vars-flt_pigname-final.txt
</pre>

== Additional notes ==
=== submitting using grid-engine ===
Submitting jobs using the grid-engine (qsub) requires a few additional options. The following lines can be added to the script to be submitted:
<pre>
#!/bin/bash
#$ -cwd
#$ -S /bin/sh
#$ -v MOSAIK_TMP=/srv/nfs02/yourhomedir/
</pre>

=== Tidying up ===
Please clean up the intermediate files as soon as they are not needed any more.
<pre>
rm *.dat
rm reads.bam
</pre>