detection of in/del in sanger sequencing

Question

[Amirosein]

Hi I'm working on Sanger sequencing reads to detect the variants and annotate them, i have only one Sanger read from specific regions (exon in example) of the genome of the patient, using sangerseqR package in R to work with it. in some patients sanger data, one of the two copies of the region have some indel so bases shift and signal start to have two peaks. along with some mismatches how to fix the data and find indel variants?

example :

from Mom: A C G T A C G T
from Dad: A C G - A C G T

so the sanger will report:

ACGTACGT
ACGACGTT

and from the 4th base there will be two peaks and basecaller may report this instead:

ACGACGGT
ACGTACTT

any idea to solve this? is there any package or function to handle that? any software who report indels? thanks all

[lh3]

Try polyphred. I used to work on a similar tool ~14 years ago in BGI, but it never got it good enough.

[Amirosein]

as i know, Polyphred could not handle Indels but only handle Substitutions.

[lh3]

As I remember, it did, but I could be wrong – haven't used it for 10+ years.

Answer 1

[Devon Ryan]

You don't really fix this in software, you sequence with primers in both directions so you can approach the indel from both sides.

[Amirosein]

in some examples, patients have some indel at the beginning and some indel at ending bases so having both directions won't help that much, i designed my own algorithm but i'm searching if there's another idea or tool o compare with.

thanks

[Amirosein]

and also enabling to fix and handle this issue will let us pay less sequencing both directions,

[Devon Ryan]

So use internal primers. Sanger sequencing isn't really designed in a way to meaningfully allow reading over indels.

[Amirosein]

ok thanks, i'll check again :)