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Sequencing issue
Hi everyone,
As suggested by Dr. Mike Trizna I am posting my query as discussion point, so that everybody can see each others comments.
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
As suggested by Dr. Mike Trizna I am posting my query as discussion point, so that everybody can see each others comments.
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
Views: 10
Replies to This Discussion
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Permalink Reply by David E. Schindel on -
I've started a discussion forum for Contig Assembly in the Managing Data discussion category. I'm posting a copy of this question there because this is a general issue of interest to all barcoders, not just botanists. I'm hoping that we'll see responses to it soon.
David -
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Permalink Reply by Tushar on -
Dear Rasika
Have you sent your sample to some commercial organization or you have sequenced it yourself? The problem you are indicating is a very "TYPICAL" problem in DNA sequencing. Due to repeat of a single base (as in AA) or repeat of a pair of bases such as GC, the detector in sequencer is not able to read 3 peaks of a single base but takes it as two. To overcome this certain software are used which normalizes the signal. So I would advice you to take this thing into account. -
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Permalink Reply by Rasika Bhagwat on -
Dear Tushar
Thanks for your reply. In my sequences the problem is not because of the repeatation of single base. The peaks of every base are very clear, but the problem is fwd and reverse sequence has difference. For example if fwd sequence has CGC sequence at particular position then reverse compliment of the reverse sequence of same sample should have the same sequence at that same position but there it will show small change like CCG, but the peaks are very clear. -
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Permalink Reply by Gaikwad Swapnil Sopan on -
Dear Rasika,
I am not able to figure out this problem. but I think this might be due to inability to detect signal by detector....How often do you encountered this problem? -
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Permalink Reply by Vincent Mulholland on -
Dear Rasika,
You might want to have a look at Zaranek, A.W. et al. A survey of genomic traces reveals a common sequencing error, RNA editing, and DNA editing. PLoS Genet. 6, e1000954 (2010) (http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal....). It shows a systematic error in runs of As. Particularly note figure 2, and associated comments in the text.
Hope this helps.
Vince -
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