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How to join two sequencing files taken form forward and reverse primer

Sequencing Forward Primer Reverse Primer

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3 replies to this topic

#1 sultan



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Posted 17 November 2012 - 11:42 PM

Hello to all,
I am new to sequencing field. I have a gene that is inserted in PCR 3.1 vector between T7 promoter and BGH terminator. My gene size is 1.8 kb. I have sequenced the vector with T7 promoter forward primer and with BGH reverse primer (both have given more than 1000 quality sequence read). I have got two sequencing files. Can any body help me that how can I get join the the results from both forward and reverse primer to get the complete sequence of the gene.
Thanking in anticipation.

#2 John Forsberg

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Posted 18 November 2012 - 07:51 AM

In our lab we use Sequencher for aligning our sequences because our university has a site license for it, but you can align the sequences without purchasing software by putting your non-coding strand in a reverse-complement calculator (like http://www.bioinform...s/rev_comp.html). Then you can use the output in any sequence alignment site you want (BLAST or ClustalW or whatever, although they may handle converting to reverse complement themselves; I didn't check), or you can just look at your forward sequence, find a series of 8 residues or so and search for them in the second sequence. Once you know how they match up, you just have to copy and paste the extra sequence from your reverse primer reaction onto the end of your forward sequence.

Does that make sense? I'm sure there are good sequence alignment sites out there that would do this for you, but I'm not familiar with them.

Edited by John Forsberg, 18 November 2012 - 07:52 AM.

#3 sultan



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Posted 18 November 2012 - 08:56 AM

Thank you very much dear it really helped me a lot :)

#4 Trof


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Posted 18 November 2012 - 09:37 AM

Yes, you don't need to reverse anything for BLAST2seq, it will find the sequence even in reverse orientation. ClustalW AFAIK not.

(but any sequence viewing software like Chromas or FinchTV should be able to reverse the whole plot including sequence, this is generaly not only good for searching within sequence, but also if you want to print sequencing plot from reverse primer in forward orientation)

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