Jumping the green wall: the use of PNA-DNA clamps to enhance
microbiome sampling depth in wildlife microbiome research
Luis Víquez-R1*, Ramona Fleischer1,
Kerstin Wilhelm1, Marco
Tschapka1,2,,and Simone
Sommer1
1. Institute for Evolutionary Ecology and Conservation Genomics,
University of Ulm, Ulm, Germany;
2. Smithsonian Tropical Research Institute, Balboa, Ancón, Panamá
Running Headline: PNA-DNA clamps for wildlife microbiome research
Correspondence author: Luis Víquez-R
Correspondence author email:luis.viquez@alumni.uni-ulm.de
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.