16S library preparation - multiple bands

[pig_raffles]

Hi,

I am carrying out test PCRs for 16S amplicon sequencing on Illumina MiSeq. I am using standard V4 primers: 515F/806R. The aim is to profile the microbiome of fish guts under different experimental treatments.

Gels loaded with product from these test PCRs show two clear bands (see attached photo). The smaller band at 400 bp is what I would expect for the fragment size of the V4 region plus attached primers. The larger and often brighter band is ca. 600 bp in size. This is not likely due to external contamination as both the negative and positive controls (Zymo mock community) did not show this band. Negative controls showing no band and the positive only the expected 400 bp band.

Has anyone seen similar sized band in their 16S V4 PCRs? Could this be amplification of fish mitochondrial 16S?

Does anyone have advice on how to control for this non-specific band during PCR and/or how to identify it? I don't want to waste sequencing coverage on a potential PCR artefact.

Thanks in advance,

Alan

[pig_raffles]

To follow-up on my previous question, we TA cloned and then Sanger sequenced the 600 bp fragment. Blast searches of the sequenced fragment came out with the 18S sequence of a congener as the top hit.

Comparison of the 16S V4 primer sequences with the 18S sequence, reveals a high degree of overlap. So, I think we can, with a degree of certainty, say that the 600 bp band was caused by non-specific amplification of our host species' 18S. Whilst these 18S sequences would be removed during our bioinformatic analysis pipeline, we would rather not waste sequencing 'effort' on this fragment. So the next step will be to develop a blocking primer for 18S, preventing its amplification during PCR.