Contig Assembly

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Permalink Reply by David E. Schindel on December 23, 2009 at 10:07am

Posted by Rasika Bhagwat on December 4, 2009 at 3:01am in Plants

I am working on DNA barcoding of plants. I am using rpoC region and while editing sequences I have encountered with a situation where forward and reverse sequence shows 1 or 2 bases variation at same position for eg all samples with the forward primer shows AA and GCG while with reverse all samples shows AAA and GGC, respectively. This creates a problem while combining the forward and reverse sequence. Is it usual to get such kind of sequence variation with forward and reverse primer of same locus and same sample?

If yes then which one is to be correct sequence and can use for further analysis?

Thanking you

Cheers
Rasika

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Permalink Reply by Lee Weigt on February 10, 2010 at 5:41pm

are the two sequences in opposite directions of equal quality in these areas?
My suspicion would be that one of the reads is of poor quality.
Lee

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Permalink Reply by Diego Pignataro on February 26, 2010 at 11:51am

I am agree with Lee, did you check the quality for only your fw seq (for all the other same samples) and the same for rv seqs?..did u compare with other data bases (if there are available? cheers

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Permalink Reply by Rasika Bhagwat on June 16, 2010 at 8:20am

Dear Lee Weigt and Diego Pignataro:

Thanks for your reply and I am very sorry for the late response.
The answer for your question is that the quality is not always equal in opposite direction both the cases. So for that, yes, one of the reads may have poor quality but its always forward sequence versus reverse sequence in my case. Either forward or reverse will have one base extra. So I am not able to understand why its always forward vs. reverse irrespective of sequence quality?

We also tried looking at the databases but we didnt find the sequences for our plant system i.e. Dalbergia. However, we checked for the other plants from same family (Fabaceae) and sequence showed the variation but I think that may be because of difference in plant system.

Cheers
Rasika

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