Question: 16S Sanger single end read assembly
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gravatar for nilus1432
13 months ago by
nilus143230
Singapore
nilus143230 wrote:

Hi, I have a single-end 16S Sanger sequencing generated using two primers (27F and 785F). I want to identify bacterial strain using this data. How to analyse this data? My initial attempts, I tried to merge read from two primers but there are no overlapping sequences. So, 1. Should I take a union of these two reads and do search to get hits? or 2. Individually search using each read and see what are the hits? Thank you.

ADD COMMENTlink written 13 months ago by nilus143230

27F seems to be a common primer but there is no mention of 785F. Both of those are forward primers so it seems logical that your reads don't overlap. Since this is sanger you probably have only a few reads. Have you tried to just blast them at NCBI?

ADD REPLYlink modified 13 months ago • written 13 months ago by genomax92k

I don't think it makes any difference in the end whether you do one search (I assume by union you mean concatenated sequences) or two individual searches. Assuming your individual sequences are long enough to give a match of good size, it should be pretty obvious what you have there after blasting them against NT database. Honestly, I think it is easier and faster to do that and see what happens rather than take time to type a question.

ADD REPLYlink written 13 months ago by Mensur Dlakic7.2k

What did you do? How did you proccess the reads? Did you quality-trim your reads? Good Sanger sequencing reads reach 700-900bp, but often times the end of the reads is of lower quality, so even if the read goes to 800-900bp, the 27F it will not overlap the 785F read due to sequencing errors.

If you want the (near) full 16S sequence, you should sequence also using the reverse primers.

ADD REPLYlink written 13 months ago by h.mon31k

After quality trimming the read size is much smaller in certain cases. I think a way is to do search individual read wise before and after trimming and compare results.

ADD REPLYlink written 13 months ago by nilus143230
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