How to compare Sanger Sequenced data to the reference sequence?

Question

[anikcropscience]

Hello, I have a question related to analyzing Sanger sequenced data. The reference gene is >7 kb long and it has 4 CDS (1900 bp). The gene has 8 allelic forms (1900 bp). My goal is to re-sequence the 4 exons from my material and compare the allelic diversity (if there is any) with the reference alleles. I have Sanger sequenced all my materials and obtained 4 exons for each genotype. My question is how can I start analyzing this data and compare it against the reference alleles? Which software should I use and what should be my approach? Can someone please give me a headstart? Thank you very much.

Regards Anik

Answer 1

[trausch]

We developed open-source web applications to align Sanger chromatogram traces and call variants with respect to a reference genome. You can also do a reference-guided assembly:

Alignment: https://www.gear-genomics.com/sage/

Variant Calling: https://www.gear-genomics.com/indigo/

Reference-guided assembly: https://www.gear-genomics.com/pearl/

[anikcropscience]

Oh, that is cool. But it does not have the desired reference sequence. I can download and upload the ref. seq. though. thank you.

[trausch]

If it is a commonly used model organism we can add the reference sequence to our set of pre-indexed genomes. If you are only interested in a single gene or exon the FASTA upload is indeed best.

[anikcropscience]

It is a commonly used model plant (wheat), but the gene of interest is not useful for daily research. So, I would use the fast file.

Answer 2

[GenoMax]

Take a look at MEGA (LINK), if you are not command line savvy. If you are then there are additional multiple sequence alignment programs that would be accessible.

[anikcropscience]

Thank you. No, I am not a command line expert.

[GenoMax]

Then MEGA would be a good place to start. They have tutorials/online help. Take a look at that first.

[anikcropscience]

Yes. Thank you very much for your help.