Question: How do I find the sequence between a Forward and Reverse primers in NGS when the sequence is highly variable?
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2.7 years ago by
ashleywurth100 wrote:

I recently did a run on a MiSeq NGS platform (250 bp pair end reads) for several samples/individuals. I ran each animal with a unique barcode (as a unique sample). I am interested in the actual genotype of each animal as I am seeing if there is a correlation between the genotype and a phenotype. When I do my analyses for mapping the other regions I can map to the reference genome, I merge my paired ends reads to find my SNPs. I used Galaxy BWA-MEM and visualized in IGV to find the SNPs that I was interested in with no issues. The problem is that I also included a region where between the F/R primers is a highly variable sequence to see if it would sequence in NGS. I do not know how to get the sequence for each individual as the length and nucleotides are different depending on the allele and so I do not think it can map to my reference genome (at least not that I have been able to find). This is also not a standard microsatellite where there is a consistent repeated region (ex- AG a certain number of times) or even interrupted micro satellites (ex- AG AG CT AG AG AG). It is a fairly long region (396-500). Is there a tool to pull out the sequence in between a set of primers? Or is this not possible with NGS reads?

sequence next-gen • 538 views
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