16S rRNA merge forward and reverse primers

Question

[ddowlin]

Hi all,

I have received 16S rRNA sequencing data for ~ 100 samples.

I want to use these sequences to identify the samples to genus level using the NCBI BLAST 16s bacteria and archaea database.

For each sample I have a forward primer fasta sequence (7F) and a reverse primer fasta sequence (1540R).

For ~40 I have a 'spliced' sequence which contains a fasta with a merged forward and reverse primer data. This was provided by the sequencing company. ~60 samples do not have a 'spliced' sequence.

My questions are:

1.) Is there a best practice protocol for merging forward and reverse primers?

2.) Can I concatenate the forward and reverse primer sequences into a single fasta file and search using these, or will this introduce biases?

3.) For the sequences that were not spliced, should I favour one of the two primers for identification purposes?

Many thanks!

d

[andres.firrincieli]

For each sample I have a forward primer fasta sequence (7F) and a reverse primer fasta sequence (1540R).

Is this a Sanger sequencing from a 16S rRNA clone library?

[ddowlin]

No, this was Illumina sequencing.

Answer 1

[Mensur Dlakic]

I am not sure that I understand everything you have done, so take my answers with a grain of salt. For example, are these pure culture or metagenomic samples?

1.) Is there a best practice protocol for merging forward and reverse primers?

I think you mean here merging results of forward and reverse amplicons (sequencing results). They need to have a significant overlap in order to be merged. Ordinarily it would be enough to have an overlap of maybe 20-40 bases, but 16S sequences are very similar even between different species. That's why you may need longer overlap than that - I would ask the company how they did it.

2.) Can I concatenate the forward and reverse primer sequences into a single fasta file and search using these, or will this introduce biases?

Only if you are absolutely certain that you have sequenced a pure culture sample, and if you know that your species has a single 16S rRNA. In such a case using a concatenated sequence may give you a cleaner result, but don't forget to reverse-transcribe one of them so you have the same strand.

3.) For the sequences that were not spliced, should I favour one of the two primers for identification purposes?

See above about the pure culture. Either way, I think both sequences will provide some information.

Answer 2

[noodle]

I want to use these sequences to identify the samples to genus level using the NCBI BLAST 16s bacteria and archaea database.

Release the kraken!