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Jumping the green wall: the use of PNA-DNA clamps to enhance microbiome sampling depth in wildlife microbiome research
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  • Luis Viquez-R,
  • Ramona Fleischer,
  • Kerstin Wilhelm,
  • Marco Tschapka,
  • Simone Sommer
Luis Viquez-R
University of Ulm
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Ramona Fleischer
University of Ulm
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Kerstin Wilhelm
University of Ulm
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Marco Tschapka
University of Ulm
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Simone Sommer
University of Ulm
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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.

Peer review status:ACCEPTED

18 Jun 2020Submitted to Ecology and Evolution
20 Jun 2020Submission Checks Completed
20 Jun 2020Assigned to Editor
23 Jun 2020Reviewer(s) Assigned
10 Jul 2020Review(s) Completed, Editorial Evaluation Pending
14 Jul 2020Editorial Decision: Revise Minor
12 Aug 20201st Revision Received
13 Aug 2020Submission Checks Completed
13 Aug 2020Assigned to Editor
13 Aug 2020Review(s) Completed, Editorial Evaluation Pending
24 Aug 2020Editorial Decision: Accept