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Old 02-12-2021, 04:19 PM   #1
Location: Hawaii, USA

Join Date: Feb 2012
Posts: 20
Default 16S library preparation - multiple bands


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,

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