Variant calling for sanger reads
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5.2 years ago
Amirosein ▴ 70

Hello all i have a Sanger read (an *.ab1 file which i did basecalling for it) and a reference. i want to find homo and hetero events for this sample (read) for further computations like annotating individual variants. now i did it myself but it seems there is a standard pipeline for this purpose for NGS reads. is there any pipeline for sanger reads as well? or i need to convert my read into fastq for this purpose? and what if "yes" what is the pipeline? i'm using Rstudio Bioconductor.

thanks all

variantcalling variants sanger alignment • 2.9k views
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5.2 years ago

Back in the day we used to use GAP4. Its not quite what you'd call "automated", but it makes a start. With sanger sequencing, because of the low coverage, you need to examine the sequence traces at any suspected SNPs yourself, or at least you did last time I did it.

Another free tool to do this was ExoTrace.

I don't know if either of these are still available.

I've also used software called MutationSurveyor, which is apparently still available, but isn't free.

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5.2 years ago

It does not look to me like variant calling from .ab1 files has been implemented in any publicly-available R/Bioconductor packages. The one sanger popular sanger package will perform basecalling and alignment, but stops short of variant calling: https://www.bioconductor.org/packages/devel/bioc/vignettes/sangerseqR/inst/doc/sangerseq_walkthrough.pdf

If you can step away from R, AutoCSA looks like it would meet your needs. It reads from .ab1 files directly, is free, java-based, and it looks like it can be automated to be included in a pipeline.

Also, novoSNP is a GUI-based desktop application that might be a useful reference.

There are some useful references also in this thread: Is there a way to access the data stored in a .ab1 file ?

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