Long‐range PCR allows sequencing of mitochondrial genomes from environmental DNA

Abstract

Abstract1. As environmental DNA (eDNA) from macro-organisms is often assumed to be highly degraded, current eDNA assays target small DNA fragments to estimate species richness by metabarcoding. A limitation of this approach is the inherent lack of unique species-specific single-nucleotide polymorphisms available for unequivocal species identification.2. We designed a novel primer pair capable of amplifying whole mitochondrial genomes and evaluated it in silico for a wide range of ray-finned fishes (Class: Actinopterygii). We tested the primer pair using long-range PCR and Illumina sequencing in vitro on a mock community of fish species assembled from pooling genomic DNA extracted from tissues. In situ we utilized long-range PCR and Illumina sequencing to generate fragments between 16 and 17 kb from eDNA extracted from filtered water samples. Water samples were sourced from a mesocosm experiment and from a natural stream.3. We validated our method in silico for 61 orders of Actinopterygii; we successfully sequenced mitogenomes in vitro from all six species in our mock community. In situ we recovered mitogenomes for all species present in our mesocosms. We additionally recovered mitogenomes from 10 of 12 species caught at the time of water sampling and two species previously only detected from eDNA metabarcoding of short DNA fragments from a natural stream.4. Successful amplification of large fragments (>16 kb) from eDNA demonstrates that not all eDNA is highly degraded. Sequencing whole mitogenomes from filtered water samples will alleviate many problems associated with identification of species from short-fragment PCR amplicon-based methods. K E Y W O R D SActinopterygii, eDNA, mitogenome sequencingThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

iDNA at Sea: Recovery of Whale Shark (Rhincodon typus) Mitochondrial DNA Sequences from the Whale Shark Copepod (Pandarus rhincodonicus) Confirms Global Population Structure

“…Efficiency of the recovery of whale shark mitogenomes from copepod tissue samples might be improved by sequencing gDNA extracted from the part of the copepod body that contains higher concentrations of whale shark tissue. In addition, given the intactness of whale shark gDNA (>1,000 bp) that was present in P. rhincodonicus, long range PCR of additional mitogenome regions followed by high throughput amplicon sequencing could also be explored to dramatically increase the coverage of the whale shark mitogenome for population genetic and phylogeographic applications (Deiner et al, 2017;Pavlova et al, 2017). Given that mtDNA only represents a fraction of the evolutionary history of a species due to its strict maternal inheritance and small genome size (16 kb), studies of the population genetics of whale sharks may benefit from the combination of both mtDNA and nuclear data FIGURE 4 | The mitogenome of Rhincodon typus (upper photo-note copepod parasites Pandarus rhincodonicus visible as black dots near the center of the upper lip) recovered from the shotgun sequencing of the parasitic copepod, P. rhincodonicus (lower photo).…”

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

“…It also allows for long range PCR amplification of whole mitochondrial genomes from eDNA samples (Deiner et al 2017). Bead milling lysis has been shown to cause shearing of nucleic acids, resulting in low molecular weight of extracted DNA (Bürgmann et al 2001).…”

Toward an ecoregion scale evaluation of eDNA metabarcoding primers: A case study for the freshwater fish biodiversity of the Murray–Darling Basin (Australia)

“…First, a positive relationship exists between the length of the internally amplified barcode and its taxonomic resolution power (Coissac et al., ; Meusnier et al., ) but the ability to recover DNA from environmental samples can be negatively impacted by the size of the DNA fragments (Deagle, Eveson, & Jarman, ; Jo et al., ). However, a number of recent studies have shown that this may be less problematic for eDNA derived from water samples (Bylemans, Furlan, Gleeson, Hardy, & Duncan, ; Deiner et al., ; Piggott, ). Second, reducing primer‐template mismatches can minimize biases arising from the PCR amplification but may inadvertently decrease the specificity of the primers to the taxonomic group of interest as these primers are more likely to bind to highly conserved regions (Pinol, Mir, Gomez‐Polo, & Agusti, ).…”