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Jumping the green wall: the use of PNA-DNA clamps to enhance microbiome sampling depth in wildlife microbiome research

Citation

Viquez-R, Luis et al. (2021), Jumping the green wall: the use of PNA-DNA clamps to enhance microbiome sampling depth in wildlife microbiome research, Dryad, Dataset, https://doi.org/10.5061/dryad.4tmpg4f7j

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.

Methods

Samples were colelcted in the field directly from the animals and stored in DNA shield at -20 Celsius. Total DNA was extracted and amplifications (V4 region of 16S rRNA. primers 515F/806R) with and without the PNA-DNA clamps (mPNA and cpPNA, PNA BIO Inc)  were used according to the sample treatment. Sequencing was conducted using Illumina MiSeq (2*250 cycles) and post sequence processing was done using QIIME 2 and Phyloseq. Consult the paper for more on the methods and the clamp sequences.

Usage Notes

Funding

Ministerio de Ciencia Tecnología y Telecomunicaciones (MICITT) y Consejo Nacional para Investigaciones Científicas y Tecnológicas (CONICIT), Award: FI-190B-14

The Rufford Foundation, Award: RSG 18771-1

Columbus Zoo and Aquarium

Elisabeth Kalko Stiftung, Award: 1601

Elisabeth Kalko Stiftung, Award: 1601