Question: detection of in/del in sanger sequencing
0
gravatar for Amirosein
3.2 years ago by
Amirosein70
UBC, Vancouver, CA.
Amirosein70 wrote:

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

variant detection sanger indel • 1.3k views
ADD COMMENTlink modified 3.2 years ago by Devon Ryan90k • written 3.2 years ago by Amirosein70

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

ADD REPLYlink written 3.2 years ago by lh331k

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

ADD REPLYlink written 3.2 years ago by Amirosein70

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

ADD REPLYlink written 3.2 years ago by lh331k
0
gravatar for Devon Ryan
3.2 years ago by
Devon Ryan90k
Freiburg, Germany
Devon Ryan90k wrote:

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

ADD COMMENTlink written 3.2 years ago by Devon Ryan90k

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

ADD REPLYlink written 3.2 years ago by Amirosein70

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

ADD REPLYlink written 3.2 years ago by Amirosein70

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

ADD REPLYlink written 3.2 years ago by Devon Ryan90k

ok thanks, i'll check again :)

ADD REPLYlink written 3.1 years ago by Amirosein70
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