16S library preparation - multiple bands

[pig_raffles]

Hi,

I am carrying out trial PCRs for 16S amplicon sequencing on Illumina MiSeq. We are targeting the V4 region of 16S using standard 515F/806R primers. Our aim is to profile the microbiome of fish guts under different experimental conditions.

Our starting PCR protocol is one used by an established high-throughput lab but this generates two distinct bands in certain of our samples (see attached photo). The smaller fragment is ca. 400 bp, which we would expect for the V4 region plus attached primers. The larger and often brighter band is around 600 bp, much bigger than our targeted fragment.

This larger fragment is not coming from external contamination as both our negative controls and our positive controls (Zymo mock community) do not show this band. The negative controls showing no bands and the positive only the targeted 400 bp band.

Has anyone seen similar sized bands in their 16S V4 gels? Could it be fish mitochondrial 16S amplification? Would it be 200 bp larger than standard microbial 16S V4?

Does anyone have advice on how to identify this band and/or how to best optimise my library prep. protocol to remove it, as I don't want to waste sequencing coverage on this artefact. I have tried increasing the annealing temperature and reducing the number of cycles with some positive effect.

Thanks in advance,

Alan