Chromatogram analysis and interpretation regarding homozygosity

Question

[Bioinf/Biotech]

Hello,

I am not sure if this is the correct place to post, but I hope so!

I am a student and currently learning various biotech/bioinf methods and your help would be greatly appreciated.

First question, when sequencing a part of genomic DNA, do we use dsDNA or ssDNA sample? Logically, I assume we would need to amplify only one strand using PCR, because otherwise we would get a lot of noise from SNPs?

Also, I came across a question that asks whether someone is heterozygous/homozygous judging from a chromatogram...However, chromatogram gives only one sequence data, how can we determine if someone is heterozygous or not purely from chromatogram?

I would be very grateful to anyone who answers!

Answer 1

[Devon Ryan]

You mention chromatograms, so I'm going to assume that you mean Sanger sequencing, rather than NGS (though the answer is essentially the same).

You would end up loading double stranded DNA into the wells of your plate/strip for sequencing (PCR will amplify both strands since you'll have forward and reverse primers). You would, however, put in a single primer, which will only bind to one strand, in the sequencing wells. The resulting sequencing will be of a single strand and start a bit downstream of the primer.

Sanger sequencings isn't NGS, a chromatogram represents the average of the genome sampled. If a SNP is heterozygous, you'll see peaks for both alleles (they'll be shorter than homozygous peaks...you should be able to figure out why this is).

In general, it's helpful to picture what's actually happening in the tubes at the various steps (draw it out if that helps).

[Bioinf/Biotech]

Thank you very much for answering!