Recently I sent amplicons generated from isolates of bacteria for sequencing. The sequences received (forward and reverse) are not combined and blasting of these sequences need to be performed. Which method can I use to combine the forward and reverse sequence of each of the isolate for BLAST analysis to be done?
To combine F and R sequence (Contig) you can use software such as ChromasPro, Snap Gene, Gene Edit, etc. After that, you can proceed to do BLAST
Good Luck
Teh Exodus Akwa
You are asking two questions in one. First question is how to merge sequences and second is how to blast.
- The answer to the second question is obvious. If you have the merged sequence, you can use it for whatever you want to do with it.
- Now, how to merge the sequence. Looks like you are having sequences from Sanger sequencing. So, to merge the sequences, first you need to be sure about the bases in sequences. generally one need to trim the ends of the sequences and check the bases based on chromatograms.
- After quality control, you need to convert reverse sequence to plus strand. For that, reverse compliment it.
- Now align the sequences (forward & reverse complemented). Alignment will result in an overlap. This overlap will then can be used to make a consensus sequence = Merged sequence.
- Software recommendations: Unipro Ugene.
For many sequences, this might be helpful:
I hope this web based program is the thing you need. just select your forward and reverse read file (.ab1), tick the read you want to reverse complement, assign your quality parameter and click on merge sequence. You must be able to download the merged sequences in a zipped file. This zipped file contains merged sequence and all the background information one need to know about what happened in merging process. http://hvdr.bioinf.wits.ac.za/fmt/
If I may ask, which software do you use to do your alignment? As Singh rightly said, reverse compliment the reverse sequence, use the abi file instead of the fasta file so that you can look at the quality of the electropherogram to verify the authencity of your sequence.
After the alignment, you can then get the consensus sequence and blast in NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Good luck
Both forward and reverse assemble using MEGA or chromatogram or DNA Baser. then convert into fasta file. With the fast file, you can perform blast against database of your interest.
- Badges
- Science method