Tackling critical parameters in metazoan meta-barcoding experiments: a preliminary study based on coxI DNA barcode

Abstract

Nowadays DNA meta-barcoding is a powerful instrument capable of quickly discovering the biodiversity of an environmental sample by integrating the DNA barcoding approach with High Throughput Sequencing technologies. It mainly consists of the parallel reading of informative genomic fragment/s able to discriminate living entities. Although this approach has been widely studied, it still needs optimization in some necessary steps requested in its advanced accomplishment. A fundamental element concerns the standardization of bioinformatic analyses pipelines. The aim of the present study was to underline a number of critical parameters of laboratory material preparation and taxonomic assignment pipelines in DNA meta-barcoding experiments using the cytochrome oxidase subunit-I (coxI) barcode region, known as a suitable molecular marker for animal species identification. We compared nine taxonomic assignment pipelines, including a custom in-house method, based on Hidden Markov Models. Moreover, we evaluated the potential influence of universal primers amplification bias in qPCR, as well as the correlation between GC content with taxonomic assignment results. The pipelines were tested on a community of known terrestrial invertebrates collected by pitfall traps from a chestnut forest in Italy. Although the present analysis was not exhaustive and needs additional investigation, our results suggest some potential improvements in laboratory material preparation and the introduction of additional parameters in taxonomic assignment pipelines. These include the correct setup of OTU clustering threshold, the calibration of GC content affecting sequencing quality and taxonomic classification, as well as the evaluation of PCR primers amplification bias on the final biodiversity pattern. Thus, careful attention and further validation/optimization of the above-mentioned variables would be required in a DNA meta-barcoding experimental routine.

Food Tracking Perspective: DNA Metabarcoding to Identify Plant Composition in Complex and Processed Food Products

“…Noteworthy, we observed the same behavior in the Food Supplement sample, considering the reads assigned to Taraxacum officinale (99.2%), compared to those assigned to the other taxa, and this could be explained also with primer competition effect. HTS is a powerful tool to detect various sources of DNA in a single analysis, trace quantity of foreign species DNA [34], but occurrence of PCR amplification bias [35] may cause inaccurate estimation of species composition and misleading results. Nevertheless, in the case of a mock herbal mixture, even if there was not a perfect linear relationship between weight and number of reads, the observed correlation coefficient outlined a certain degree of relationship between observed and expected frequencies (r = 0.75, p -value < 0.001).…”

“…As already noted in our results on processed food (see in the previous paragraph the case of Taraxacum officinale ), amplicon metabarcoding can be affected by the PCR amplification step using “universal” markers. The occurrence of a bias during PCR amplification may cause the inaccurate estimation of quantities and this was at least partially demonstrated for metazoan and plants [17,35,36]. This bias generates a variable number of template–primer mismatches across species, resulting in a final amplified DNA mixture that does not always reflect the original proportion of each species, limiting the quantitative potential of DNA metabarcoding [37,38,39,40].…”

COI and ND1 as DNA barcodes to identify Taenia hydatigena (Cestoda: Taeniidae) from canine small intestine on Hainan Island in China

“…The application of the barcode of the COI gene in the study of molecular biodiversity is increasing rapidly and continuously (Balech et al 2018). There are approximately 4.5 million COI gene barcodes in GenBank and BOLD (Barcode of Life Data Systems), including different individuals across 250,000 species.…”