Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale

Abstract

Background In light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). Findings Using a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed Nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. Conclusions Sequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost-effective, portable, and universal approach for eukaryote DNA barcoding. Although bulk community analyses using long-amplicon approaches may introduce biases, the long rDNA amplicons approach signifies a powerful tool for enabling the accurate recovery of taxonomic and phylogenetic diversity across biological communities.

Reappraising plastid markers of the red algae for phylogenetic community ecology in the genomic era

“…Moreover, if the target genes are too long for short-read HTS technologies by Illumina Inc., long-read sequencing technologies, such as the MinION by Oxford Nanopore Technologies Ltd., provide a promising alternative approach. The handheld, affordable, and field-deployable MinION boasts long sequencing read lengths of thousands to millions of base pairs long (e.g., Krehenwinkel et al, 2019). This powerful feature enables the sequencing of entire genes without the need to correct for assembly errors (i.e., chimeric sequences) (see Saunders & Moore, 2013).…”

Genomics in the jungle: using portable sequencing as a teaching tool in field courses

“…Project 4 -DNA barcoding: In this study, we used a compilation of samples of known and unknown taxonomic identities in a multiplexed reaction across a range of markers. For plant samples, we used matk, rBCL, trnh-psBA and a set of long-range rRNA primers (Krehenwinkel et al, 2019;Kress & Erickson, 2012). For mammals we used a cocktail of cytochrome oxidase 1 primers (Kress & Erickson, 2012).…”

“…We then attempted to multiplex all successful samples that had PCR amplicons verified via agarose gel electrophoresis onto a single flowcell run, as per the protocol on protocols.io . We created consensus sequences for the respective barcodes as described in Pomerantz et al (2018) and Krehenwinkel et al (2019).…”

Lead soaps formation and biodiversity in a XVIII Century wax seal coloured with minium

“…This technology offers tremendous potential for long-read barcoding applications, as recently shown in an analysis in fungi. Long amplicons scale up DNA barcoding by enabling the accurate recovery of taxonomic and phylogenetic diversity (Krehenwinkel et al, 2019).…”