BLAST result is different between Sanger forward and reverse reads
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6.2 years ago
Louis Kok ▴ 20

Hi all, Just wish to get your opinions. If the Sanger forward and reverse reads were base called separately, performed BLASTN against NCBI nt database and the result was found to be different species belonging to the same genus, how would you decide which result to take if both reads have clean peaks shown in chromatogram? Thanks a lot. 

BLAST Sanger Forward Read Reverse Read • 2.5k views
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Are they exactly the reverse complementary of each other? 

Or the forward sequence contains mainly the left part of the sequence, and the reverse one mainly the right part of the sequence ?

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Hi Antonio, using CAP3 to assemble forward and reverse reads, even with very low overlap percent identity cutoff (65%), still couldn't produce merged sequence. I suppose they are not overlapped each other. 

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You mention 65% of identity, but not the extension of that identity.. That could eventually join unrelated sequences, and therefore different sequences are found by Blast.

If you have sequenced the same samples in forward and reverse orientations, and you mention you got clean peaks, I am also wondering why you have used such as low cutoff value..

My first approach would be to increase that cutoff value with a more stringent value. CAP3 will render you singletons and contigs accordingly.

If different results are obtained, evaluate the values of E (the lower the better) and the query extension (the higher the better).  

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