DOI: 10.5061/dryad.q5gg0
title: Data from: Long-range PCR allows sequencing of mitochondrial genomes from environmental DNA

description: 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. 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. 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. Successful amplification of large fragments (&gt;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.
description: commands_used_for_analysisAll commands used to execute bioinformatic pipeline.norm_50xCommand used to down sample reads for de novo assembly.mitogenomes_all_corrected_reading_frame.v0301File contains the reference sequences used for mapping the .sam files to and is needed for importing .sam files into Geneious for SNP analysisJudayCreek.scaffoldContains all scaffolds that were assembled from the Juday Creek water samples.JudayCreek.scaffold.fa.blast.filter.summary.scafList subset of scaffolds by species that were used to map to references in Geneious and is illustrated in Figure 2 of manuscript.Mesocosm_EH.scaffoldContains all scaffolds that were assembled from the even high abundance mesocosm water samples.Mesocosm_EH.scaffold.fa.blast.filter.summary.scafList subset of scaffolds by species that were used to map to references.Mesocosm_EH.aligning.unique.sorted.MarkedDuplicatesFile contains all reads mapped to references considered in study. The file in conjunction with mitogenomes_all_corrected_reading_frame.v0301.txt allows the SNPs to be called in using Geneious.Mesocosm_SH.scaffoldContains all scaffolds that were assembled from the skewed high abundance mesocosm water samples.Mesocosm_SH.scaffold.fa.blast.filter.summary.scafList subset of scaffolds by species that were used to map to references.Mesocosm_SH.aligning.unique.sorted.MarkedDuplicatesFile contains all reads mapped to references considered in study. The file in conjunction with mitogenomes_all_corrected_reading_frame.v0301.txt allows the SNPs to be called in using Geneious.Mock.50x.scaffoldContains all scaffolds that were assembled from the mock community sample.Mock.50x.scaffold.fa.blast.filter.summary.scafList subset of scaffolds by species that were used to map to references.Mock.aligning.unique.sorted.MarkedDuplicates.samFile contains all reads mapped to references considered in study. The file in conjunction with mitogenomes_all_corrected_reading_frame.v0301.txt allows the SNPs to be called in using Geneious.JudayCreek.aligning.unique.sorted.MarkedDuplicatesZipped file contains all reads mapped to references considered in study. The file in conjunction with mitogenomes_all_corrected_reading_frame.v0301.txt allows the SNPs to be called in using Geneious and for generation of mapping results illustrated in Figure 2.