Introduction
A new world of research opportunities has emerged with the advancement of sequencing techniques. One of the fields that have benefited most is the study of whole microbial communities, so-called microbiomes. This novel method allows the study of bacterial mats and gut microbiome linings without the need to cultivate each bacterium separately (Caporaso et al. 2012). Together with recently developed and improved bioinformatic pipelines (Mothur, QIIME 2, etc.), we now have the means to classify and assign taxonomy with a reasonable level of confidence (Bolyenet al.2019).
As microbiome research moves away from model organisms and extends into natural settings, new challenges of wildlife research and those arising because of the variety of wildlife diets need to be tackled. One of the drawbacks is that the separation of bacterial sequences from non-bacterial signals, i.e. those from mitochondria (from the host) and chloroplasts (from the diet) can sometimes be tricky (Barottet al. 2011; Lundberg et al.2013). According to the widely accepted endosymbiosis theory (Margulis [then known as Sagan (1967), Gray 2017], mitochondria and chloroplasts were originally derived during evolution from hijacked bacteria engulfed by other bacteria. Because of this bacterial origin, some DNA sequences of organelles are strikingly bacteria-like. This is also the case with reads obtained from high throughput sequencing of 16S rRNA genes, the usual target of microbiome studies. In the worst case, the resulting read coverage consists of many reads assigned to mitochondria or chloroplasts.
Several ways are available to circumvent this problem; the most common path is to increase the sequencing depth and then filter out the reads assigned to the organelles. However, this technique results in an expensive price tag for sequencing and may lead to highly skewed read numbers depending on the provenance of the samples. Another option has recently arisen: the use of DNA-PNA clamps as PCR blockers to prevent the amplification of the specific mitochondrial or chloroplast sequences (Fitzpatricket al. 2018). PNAs (peptide nucleic acids) are DNA-mimicking molecules with outstanding hybridization properties (Nielsen & Egholm 1999). The backbone of the molecules is constructed of N-(2-amino-ethyl) glycyl (AEG) instead of the sugar-phosphate backbone of DNA (Nielsen, Egholm & Buchardt 1994). The nucleobases attached to this backbone are the same as those in DNA, thereby allowing hybridization between the probe and the bacterial DNA. PNAs are thus a powerful molecular tool in microbiome research for dealing with samples with a high content of either host or plant remnants in faecal pellets (Lundberg et al. 2013).
In this study, we tested the PNA-DNA clamps as a method for improving microbiome discovery rates in bats (Tequila bat Leptonycteris yerbabuenae ) and Galapagos mockingbirds (Mimus parvulus ). We chose these two study organisms because they both rely on heavy plant-based diets that sometimes can lead to masses of plant-based faecal material producing high contamination signals from chloroplast 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 the unwanted sequencing reads.