Amplicon metabarcoding is an established technique to analyse the taxonomic composition of communities of organisms using high-throughput DNA sequencing, but there are doubts about its ability to quantify the relative proportions of the species, as opposed to the species list. Here, we bypass the enrichment step and avoid the PCR-bias, by directly sequencing the extracted DNA using shotgun metagenomics. This approach is common practice in prokaryotes, but not in eukaryotes, because of the low number of sequenced genomes of eukaryotic species. We tested the metagenomics approach using insect species whose genome is already sequenced and assembled to an advanced degree. We shotgun-sequenced, at low-coverage, 18 species of insects in 22 single-species and 6 mixed-species libraries and mapped the reads against 110 reference genomes of insects. We used the single-species libraries to calibrate the process of assignation of reads to species and the libraries created from species mixtures to evaluate the ability of the method to quantify the relative species abundance. Our results showed that the shotgun metagenomic method is easily able to set apart closely-related insect species, like four species of Drosophila included in the artificial libraries. However, to avoid the counting of rare misclassified reads in samples, it was necessary to use a rather stringent detection limit of 0.001, so species with a lower relative abundance are ignored. We also identified that approximately half the raw reads were informative for taxonomic purposes. Finally, using the mixed-species libraries, we showed that it was feasible to quantify with confidence the relative abundance of individual species in the mixtures.

Mitochondrial metagenomics or mito-metagenomics (hereafter, MMG) is becoming an alternative to the classical amplicon metabarcoding for the large-scale assessment of biodiversity of Metazoa

The use of high-throughput sequencing to recover short DNA reads of many species has been widely applied on biodiversity studies, either as amplicon metabarcoding or shotgun metagenomics. These reads are assigned to taxa using classifiers. However, for different reasons, the results often contain many false positives. Here we focus on the reduction of false positive species attributable to the classifiers. We benchmarked two popular classifiers, BLASTn followed by MEGAN6 (BM) and Kraken2 (K2), to analyse shotgun sequenced artificial single-species samples of insects. To reduce the number of misclassified reads, we combined the output of the two classifiers in two different ways: (1) by keeping only the reads that were attributed to the same species by both classifiers (intersection approach); and (2) by keeping the reads assigned to some species by any classifier (union approach). In addition, we applied an analytical detection limit to further reduce the number of false positives species. As expected, both metagenomic classifiers used with default parameters generated an unacceptably high number of misidentified species (tens with BM, hundreds with K2). The false positive species were not necessarily phylogenetically close, as some of them belonged to different orders of insects. The union approach failed to reduce the number of false positives, but the intersection approach got rid of most of them. The addition of an analytic detection limit of 0.001 further reduced the number to ca. 0.5 false positive species per sample. The misidentification of species by most classifiers hampers the confidence of the DNA-based methods for assessing the biodiversity of biological samples. Our approach to alleviate the problem is straightforward and significantly reduced the number of reported false positive species.