How to analyse Sanger sequence data?

Question

[anikcropscience]

Hi there, I have some confusion regarding the analysis of Sanger sequencing results. My reference gene is 7kb long and has 4 exons (coding sequence) with different lengths of intervals. Additionally, the gene has 8 alleles that are already published. My goal is to resequence all my samples and find if I can recover all the alleles or find new ones. So, I have 200 samples and I have successfully obtained the sequences of the 4 exons ONLY. I did not resequence the whole gene but the coding regions. When I align my sequences to the reference gene, there are some parts of my sequences that span over the introns. My question is: 1. Do I have to manually delete these extra parts and assemble them to make the alleles to see whether I obtain the same published alleles or different ones? 2. If I want to translate the gene and my sequences to find non-synonymous mutations, should I translate only the coding region of the gene and my sequences? or Should I translate the whole gene?

I am using Geneious software at the moment for all the alignment analyses. Below is a figure showing some alignment of my sequences to the reference gene (Yellow is the CDS and red is the mRNA). I have sequenced these red/yellow blocks.

Original picture link.

[GenoMax]

You should contact Geneious tech support (you are paying for a license) and get specific help that would directly be usable with Geneious.

That said as long as you know where the coding exons are and you have the complete reference you can align your sanger data to that. You are not going to have a ton of sanger reads so looking for alleles should be an easy enough task after doing a multiple sequence alignment of your sanger data and reference. Your alleles are single base pair changes so they should be easily discernible.

[anikcropscience]

Actually, the question is not about the use of the software. Okay, I understand your point. But what about translating the whole gene and the sequences that I obtained?

[GenoMax]

Can you clarify what did you actually sequence? Just RNA or original DNA? What are those box like structures representing (not familiar with Geneious interface)?

[anikcropscience]

This is original DNA (PCR product). Each box like structure is an alignment of one sample to the above (colored) reference gene. Here I sequenced the 3 exons of the gene indicated by those CDS/mRNA.

[GenoMax]

Please use How to add images to a Biostars post

Answer 1

[JC]

1. Do I have to manually delete these extra parts and assemble them to make the alleles to see whether I obtain the same published alleles or different ones?

No, you can call variants with them, just be sure your calls are inside the exons of your interest

1. If I want to translate the gene and my sequences to find non-synonymous mutations, should I translate only the coding region of the gene and my sequences? or Should I translate the whole gene?

You just need to be sure to be in the same frame.

Answer 2

[swbarnes2]

Just to be clear, what did you sequence off of? cDNA? DNA?

I'd note every SNP, and annotate them with something like ensembl's Variant Effect Predictor, to make clear which are coding, and which are not.

[anikcropscience]

I sequenced DNA extracted from the plant leaf and then ran PCR on them which were sent for Sanger sequencing.

[swbarnes2]

Okay, so why are you surprised that you have intronic sequence? How else would you get accurate sequence of the entire exon?