BWA MEM question

CHObot · 07-26-2013, 02:37 PM

I am using BWA MEM to map 250 bp MiSeq PE reads. I am doing targeted genomic sequencing, enriching the library for specific transgenic sequences. I make a reference sequence, map all of the reads to that reference (using the default parameters). What I get are many improperly mapped reads. Some of the reads that map have 20 - 50 mismatches between the read and the reference, but it maps them none-the-less. There are similar sequences contaminating the sample apparently, but I don't want these spurious mappings. How can I increase the stringency of the mapping, so I don't get all of these imporperly mapped reads? I have more than enough coverage to throw out any bad mappings and still have tons of coverage.

Thanks!
CHObot

P.S. I am looking for chimeric reads at the ends of the mappings to determine transgenic insertion sites. Is BWA MEM the best algorithm for this? It seems to work, albeit with a lot of manual examination of chimeric reads.

sdriscoll · 07-26-2013, 06:31 PM

you should be able to threshold the alignments by the MAPQ value. I'm not sure of the specific cutoff but let's say 20.

Code:

samtools view -bq 20 alignments.bam > filtered.bam

bwa has a very descriptive MAPQ field so this *should* work. Optionally maybe there's another tool out there that can specifically filter by mismatches if you want to keep the mismatches down to say 4 or 5% of the read length.

jp. · 08-08-2013, 02:05 AM

can some post general commands to align fq PE with hg19
where should I start with ?

mastal · 08-08-2013, 02:11 AM

the bwa manual webpage gives examples of the various bwa commands:

http://bio-bwa.sourceforge.net/bwa.shtml

jp. · 08-08-2013, 03:38 AM

I used bwa aln -t 4 -f testsample.sai /GATKbundle/ucsc.hg19.fasta /testsample.fastq
It was finished within 2 minutes and generated testsample.sai. Is this the alignment file ?
I used bowtie and it took 4-5 hrs but bwa is so fast ??? something is wrong ????

Quote:

Originally Posted by mastal

the bwa manual webpage gives examples of the various bwa commands:

http://bio-bwa.sourceforge.net/bwa.shtml

mastal · 08-08-2013, 04:04 AM

bwa aln is part of a 2-step process.

the first step generates the .sai file.

the second step is bwa samse/sampe, depending on whether you have single end or paired-end reads, and should give you a .sam file.

the manual will give you the details.

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07-26-2013, 02:37 PM	#1
CHObot Junior Member Location: Greater Boston Area Join Date: May 2013 Posts: 8	BWA MEM question I am using BWA MEM to map 250 bp MiSeq PE reads. I am doing targeted genomic sequencing, enriching the library for specific transgenic sequences. I make a reference sequence, map all of the reads to that reference (using the default parameters). What I get are many improperly mapped reads. Some of the reads that map have 20 - 50 mismatches between the read and the reference, but it maps them none-the-less. There are similar sequences contaminating the sample apparently, but I don't want these spurious mappings. How can I increase the stringency of the mapping, so I don't get all of these imporperly mapped reads? I have more than enough coverage to throw out any bad mappings and still have tons of coverage. Thanks! CHObot P.S. I am looking for chimeric reads at the ends of the mappings to determine transgenic insertion sites. Is BWA MEM the best algorithm for this? It seems to work, albeit with a lot of manual examination of chimeric reads.

07-26-2013, 06:31 PM	#2
sdriscoll I like code Location: San Diego, CA, USA Join Date: Sep 2009 Posts: 438	you should be able to threshold the alignments by the MAPQ value. I'm not sure of the specific cutoff but let's say 20. Code: samtools view -bq 20 alignments.bam > filtered.bam bwa has a very descriptive MAPQ field so this should work. Optionally maybe there's another tool out there that can specifically filter by mismatches if you want to keep the mismatches down to say 4 or 5% of the read length. __________________ /* Shawn Driscoll, Gene Expression Laboratory, Pfaff Salk Institute for Biological Studies, La Jolla, CA, USA */

08-08-2013, 02:05 AM	#3
jp. Senior Member Location: NikoNarita.jp Join Date: Jul 2013 Posts: 142	can some post general commands to align fq PE with hg19 where should I start with ?

08-08-2013, 02:11 AM	#4
mastal Senior Member Location: uk Join Date: Mar 2009 Posts: 667	the bwa manual webpage gives examples of the various bwa commands: http://bio-bwa.sourceforge.net/bwa.shtml

08-08-2013, 04:04 AM	#6
mastal Senior Member Location: uk Join Date: Mar 2009 Posts: 667	bwa aln is part of a 2-step process. the first step generates the .sai file. the second step is bwa samse/sampe, depending on whether you have single end or paired-end reads, and should give you a .sam file. the manual will give you the details.