Dear all,
I just recently started to evaluate MiSeq paired-end reads of 150bp in length for both research and diagnostic purposes. So far I used a pipeline consisting of BWA for the alignment, GATK for the SNP/INDEL calling and ANNOVAR for annotation of detected variants.
A colleague of mine used a different analysis tool for diagnostics and reported a 46bp insertion in one of our target genes (BRCA1). She also confirmed it with gel-electrophoresis and clearly two bands are visible. However, I am not able to detect this insertion with my pipeline, neither with GATK nor am I able to visualize it with IGV. Thus, I’m thinking that my alignment might be incorrect.
As I only used the default settings for BWA, I thought I can tweak some parameters. I tried playing around with the bwa aln –e and -o option but so far with no success. Additionally, I tried bowtie for the alignment but also couldn’t detect the insertion.
What could be the best parameters for BWA to detect insertions of this length? Is there a limit for the length of recognizable insertions? Or should I even use a different alignment tool?
I would be really thankful for any remarks and comments!
Martin
I just recently started to evaluate MiSeq paired-end reads of 150bp in length for both research and diagnostic purposes. So far I used a pipeline consisting of BWA for the alignment, GATK for the SNP/INDEL calling and ANNOVAR for annotation of detected variants.
A colleague of mine used a different analysis tool for diagnostics and reported a 46bp insertion in one of our target genes (BRCA1). She also confirmed it with gel-electrophoresis and clearly two bands are visible. However, I am not able to detect this insertion with my pipeline, neither with GATK nor am I able to visualize it with IGV. Thus, I’m thinking that my alignment might be incorrect.
As I only used the default settings for BWA, I thought I can tweak some parameters. I tried playing around with the bwa aln –e and -o option but so far with no success. Additionally, I tried bowtie for the alignment but also couldn’t detect the insertion.
What could be the best parameters for BWA to detect insertions of this length? Is there a limit for the length of recognizable insertions? Or should I even use a different alignment tool?
I would be really thankful for any remarks and comments!
Martin
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