Quantifying allele fraction of small to medium deletions in amplication sequencing

brentonmar · 08-07-2013, 06:37 AM

Hi all,

I would like to use MiSeq amplicon sequencing to detect the clonal outgrowth of cells harboring small to medium sized deletions (1bp to 160bp) in specific genes generated using CRISPR technology (which makes random sized deletions at specific loci matching a DNA template).

I've used BWA/Varscan in the past to quantify the allele fraction of small (1-3bp) deletions. I've also used pindel to qualitatively identify larger deletions, but I'm unsure if one can quantatively estimate the allele fraction of deletion using pindel or other tools. Has anyone tried this or have any advice?

Thanks!
-Brenton

chrisamiller · 08-08-2013, 06:56 AM

It's a challenging issue that usually requires several steps:

1) local assembly to identify the exact sequence of the large indel
2) creation of a contig containing that sequence
3) competitive realignment to discover reads that span the breakpoint, but couldn't be mapped previously

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08-07-2013, 06:37 AM	#1
brentonmar Junior Member Location: Boston Join Date: Jan 2012 Posts: 1	Quantifying allele fraction of small to medium deletions in amplication sequencing Hi all, I would like to use MiSeq amplicon sequencing to detect the clonal outgrowth of cells harboring small to medium sized deletions (1bp to 160bp) in specific genes generated using CRISPR technology (which makes random sized deletions at specific loci matching a DNA template). I've used BWA/Varscan in the past to quantify the allele fraction of small (1-3bp) deletions. I've also used pindel to qualitatively identify larger deletions, but I'm unsure if one can quantatively estimate the allele fraction of deletion using pindel or other tools. Has anyone tried this or have any advice? Thanks! -Brenton

08-08-2013, 06:56 AM	#2
chrisamiller Junior Member Location: USA Join Date: Jun 2008 Posts: 7	It's a challenging issue that usually requires several steps: 1) local assembly to identify the exact sequence of the large indel 2) creation of a contig containing that sequence 3) competitive realignment to discover reads that span the breakpoint, but couldn't be mapped previously