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Old 07-26-2013, 01:37 PM   #1
CHObot
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Default 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.
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Old 07-26-2013, 05:31 PM   #2
sdriscoll
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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.
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Old 08-08-2013, 01:05 AM   #3
jp.
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can some post general commands to align fq PE with hg19
where should I start with ?
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Old 08-08-2013, 01:11 AM   #4
mastal
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the bwa manual webpage gives examples of the various bwa commands:

http://bio-bwa.sourceforge.net/bwa.shtml
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Old 08-08-2013, 02:38 AM   #5
jp.
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Question

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 ????

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Originally Posted by mastal View Post
the bwa manual webpage gives examples of the various bwa commands:

http://bio-bwa.sourceforge.net/bwa.shtml
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Old 08-08-2013, 03:04 AM   #6
mastal
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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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