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
You could try a higher annealing temperature in PCR, perhaps using one or two of those samples and doing a gradient PCR with higher annealing temperatures. If it is an unspecific band, it should disappear.
I would also decrease the amount of DNA and/or the number of cycles. The smear often indicated excess DNA, and that can potentiate unspecific amplifications. For example, P1_E9 less smear, no unspecific.
You could try to excise the band and sequence it, although it may be a mixture and often difficult to get any good result.
About the “fish mitochondrial 16S” hypothesis, I would do an alignment with a few sequences and see if the primer binding sites are similar. And you could try to use DNA from a fish and test the primers.
Yes, you can amplify fish mitochondrial 16S with these primers. You can either try to optimize your PCR annealing conditions, cut out and sequence just the smaller PCR products, or sort out any fish sequences bioinformatically after you get your data.
Azhar Al-Ankoshy Thanks!
Katie A Burnette, Filipe Pereira Thank you both for the detailed replies. We have tried increasing the annealing temperature, which did decrease the 600 bp band and at the same time increased the brightness of the 400 bp band somewhat. Cycle number is next on our optimisation list.
As Filipe says, sequencing the non-specified band may be tricky as it may be a mix of sequences.
I am surprised that no one else has come across a similar problem given the level of interest in fish gut microbiomes and the popularity of the V4 515F/806R primer pair in the field
Maybe there is some organism in your particular microbiome sample whose DNA is being amplified in an unspecific way by those primers. Could be rare and never detected before. I have found “strange” sequences in environmental samples. You could also try a different polymerase (some are better avoiding unspecific amplifications), but you will most likely solve the issue before that. Good luck!
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