Jumping the green wall: the use of PNA-DNA clamps to enhance microbiome
sampling depth in wildlife microbiome research
Abstract
As microbiome research moves away from model organisms to wildlife, new
challenges for microbiome high throughput sequencing arise caused by the
variety of wildlife diets. High levels of contamination are commonly
observed emanating from the host (mitochondria) or diet (chloroplast).
Such high contamination levels affect the overall sequencing depth of
wildlife samples thus decreasing statistical power and leading to poor
performance in downstream analysis. We developed an amplification
protocol utilizing PNA-DNA clamps to maximize the use of resources and
to increase the sampling depth of true microbiome sequences in samples
with high levels of plastid contamination. We chose two study organisms,
a bat (Leptonyteris yerbabuenae) and a bird (Mimus parvulus), both
relying on heavy plant-based diets that sometimes lead to traces of
plant-based faecal material producing high contamination signals from
chloroplasts and mitochondria. On average, our protocol yielded a
13-fold increase in bacterial sequence amplification compared with the
standard protocol (Earth Microbiome Protocol) used in wildlife research.
For both focal species, we were able significantly to increase the
percentage of sequences available for downstream analyses after the
filtering of plastids and mitochondria. Our study presents the first
results obtained by using PNA-DNA clamps to block the PCR amplification
of chloroplast and mitochondrial DNA from the diet in the gut microbiome
of wildlife. The method involves a cost-effective molecular technique
instead of the filtering out of unwanted sequencing reads. As 33% and
26% of birds and bats, respectively, have a plant-based diet, the tool
that we present here will optimize the sequencing and analysis of wild
microbiomes.