4 replies to this topic

[pDNA]

Dear people,

maybe someone can help me get along with the following problem:

I sequenced a genomic region of an E. coli strain by nested PCR. The results so far look fine, sequencing quality is good, but there is this strange 20 bp spot within the assembled sequence that gives trace overlays. Left and right from that spot the sequence quality is fine and the base calling is exact, but within this 20 bp region there is something strange going on with that sequence. I've attached a pic of the traces ...maybe someone of you can interpret this properly.

Does it mean that there is a mixed population that differs within these 20 bp region? ...or is it possible that this is a kind of sequencing artefact due to ...whatever?

Many thanks in advance!

Regards,
p

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[phage434]

Yes, this looks exactly as if two locations in the genome both amplified and differed in that region.

[pDNA]

phage434, on Feb 15 2010, 09:50 PM, said:

Yes, this looks exactly as if two locations in the genome both amplified and differed in that region.

many thanks!

i was not sure how to deal with that ...but there must be something going within that spot.

Is there a way to dissect such trace overlays ...or do i have to do it manually?

Regards,
p

[DrAnt1]

... as you know your sequence that you're interested in, you can work out from those 20bp which are the 'wrong' base pairs ... have you run a BLAST search on those 20bp to see what they are/where they're from? http://blast.ncbi.nl...h.gov/Blast.cgi

[Warren]

pDNA, on Feb 15 2010, 03:55 PM, said:

phage434, on Feb 15 2010, 09:50 PM, said:

Yes, this looks exactly as if two locations in the genome both amplified and differed in that region.

many thanks!

i was not sure how to deal with that ...but there must be something going within that spot.

Is there a way to dissect such trace overlays ...or do i have to do it manually?

Regards,
p

Why not just clone the PCR fragment and pick a few clones to get both sequences? Should be straight forward. Plus it will verify that you actually are seeing amplification of two different regions differing in those areas, and not some other strange artifact. It may turn out to something very cool.

Warren..