16S Sanger single end read assembly
0
0
Entering edit mode
4.5 years ago
nilus1432 ▴ 30

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.

sequencing Assembly microbiome 16S • 1.5k views
ADD COMMENT
0
Entering edit mode

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 REPLY
0
Entering edit mode

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 REPLY
0
Entering edit mode

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 REPLY
0
Entering edit mode

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 REPLY

Login before adding your answer.

Traffic: 2248 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6