Can someone assist me regarding the analysis of 16sRNA sequences. After my PCR, I sent my number of samples for sequencing. I got report from them as Forward Trimmed sequence and Reverse trimmed sequence. I am following below method with Bioedit software for analyzing and getting results for it.
For example, I use Forward sequence and reverse sequence in a bioedit software.I make compliment side for reverse sequence. After I finding overlapping region , I copy any of the strands overlapping area either forward or reverse and paste it in word file. Again I copy remaining ares of the strand which would be pasted in same word file, I change it in FASTA format then I check it in NCBI blast further.
One sample here
After overlapping,
forward AAAATTTTTGGGTCTCGA
reverse TTTTTCCCAGAGCT ---->
I copy above strand AAAATTTTT and below strand CCCAGAGCT (leaving below overlapping region )and I make AAAATTTTTCCCAGAGCT as whole strand copy paste in NCBI BLAST to get bacterial name.
Am I doing it correct or is there any other way, software. Please refer to the following attachment, tell me which sample you are getting. Forward sequence attached, reverse sequence will be in next message.
As far as i remember you do it right... For quick sequence assignment use http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp
The easiest way to join the sequences:
1. Reverse complement the reverse sequence using EMBOSS revseq:
http://emboss.bioinformatics.nl/cgi-bin/emboss/revseq
2. Then find the overlap and get the joined sequence using EMBOSS union:
http://emboss.bioinformatics.nl/cgi-bin/emboss/revseq
Paste both sequences with a FASTA header each (or upload them from a file) AND change "Look for overlaps when joining?: No" to "Yes"
Then use either seqmatch (see answer above) or the classifier (for a quick first result)
https://rdp.cme.msu.edu/classifier/classifier.jsp
Hi Subash
The way you are doing contig in Bioedit, sounds little different for me.
In Bioedit by using “cap contig assembly program” you can easily do the analysis. Just follow the following steps:
1) Keep your both forward and reverse sequence in a single text file and save as “.fasta” format.
2) Now open the sequence in Bioedit (ver 7.1).
3) Click on “Accessory applications” followed by “cap contig assembly program” (let it be default settings in the dialogue box) and click on the “Run application”.
4) A command prompt window will appear. Just close it and your job is done.
5) You could be able to see an output window where the third line from the top will be your contig sequence (contig-0). You can copy that sequence and go for BLASTn analysis.
All the best :-)
If still any doubts contact me at sunil.mangroves@gmail .com
Dear Sunil,
Thank you for your effort and wonderful reply at research gate. I used it as the same way you advise.It matches what I expected. But in your explanation you mentioned that use forward and reverse sequence as a single file.
What I do here is, copy forward sequence and paste in Bioedit (using import from clipboard at File menu)and again I copy reverse sequence in similar way. I press ctrl+shift+R so it gives complementary strand for reverse sequence.( Which gives plus/plus strand position in NCBI finally)
Again I do what you suggested like
Click on “Accessory applications” followed by “cap contig assembly program”and click on the “Run application”.
A command prompt window will appear. Just close it and your job is done.
You could be able to see an output window where the third line from the top will be your contig sequence (contig-0). You can copy that sequence and go for BLASTn analysis.
Thats the way I do here , I am getting good report indicating that is plus /plus strand. I attached notepad documents which are directly from sequencing company.Please check in your free time( I copied forward and reverse sequence in single file coz here only one attachment is allowed)
I got a ncbi report for following sequences -indicating that
Pseudomonas fluorescens strain S181R 16S ribosomal RNA gene, partial sequence
Sequence ID: gb|JF513138.1| Length: 1535 Number of Matches: 1
If you also got the same report, I 'll feel happy that I did in correct way.Looking for your response.
I tried with you mail id sunil.mangroves@gmail .com
- Badges
- Science method