Field-based species identification of closely-related plants using real-time nanopore sequencing

Abstract

Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere. However, generation of datasets from large, complex genomes has been constrained to laboratories. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed. Here, we show that a field-based RTnS analysis of closely-related plant species (Arabidopsis spp.) has many advantages over laboratory-based high-throughput sequencing (HTS) methods for species level identification and phylogenomics. Samples were collected and sequenced in a single day by RTnS using a portable, “al fresco” laboratory. Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification. Individually annotated RTnS reads can be used to infer the evolutionary relationships of A. thaliana. Furthermore, hybrid genome assembly with RTnS and HTS reads substantially improved upon a genome assembled from HTS reads alone. Field-based RTnS makes real-time, rapid specimen identification and genome wide analyses possible.

Host‐derived population genomics data provides insights into bacterial and diatom composition of the killer whale skin

“…Contamination in the laboratory could be further controlled for and characterized through inclusion of extraction, library preparation and PCR blanks as negative controls (Lusk, 2014;Salter et al, 2014) and measures such as double indexing (Kircher, Sawyer, & Meyer, 2011;Rohland & Reich, 2012;van der Valk, Vezzi, Ormestad, Dalén, & Guschanski, 2018), which can then inform the emerging downstream filtering methods for separating true microbiomes from contamination (Delmont & Eren, 2016;Davis et al, 2017). Lastly, the advances in long-read sequencing using portable nanopore-based platforms make it possible to generate data suitable for reconstructing complete bacterial genomes while in the field (Parker, Helmstetter, Devey, Wilkinson, & Papadopulos, 2017), including in the Antarctic (Johnson, Zaikova, Goerlitz, Bai, & Tighe, 2017). This is a promising development with respect to improving the breadth of host taxa from which bacterial taxa are derived and should improve future mapping of metagenomics data and taxonomic assignment.…”

Global Island Monitoring Scheme (GIMS): a proposal for the long-term coordinated survey and monitoring of native island forest biota

“…Furthermore, the recent advances in sequencing techniques now allow third-generation sequencing directly in the field (e.g. Parker et al 2017), which opens very promising avenues for novel ways of DNA-based identification beyond the limits of predefined barcoding regions. In particular, given the importance of being able to assess identity and abundance of all individuals in an entire community, approaches are being developed that allow massive sequencing of entire communities at minimal cost (Krehenwinkel et al 2018).…”

Generating DNA sequence data with limited resources for molecular biology: Lessons from a barcoding project in Indonesia

“…Advances are being made in all relevant areas, including DNA amplification (e.g., isothermal PCR [Boyle et al., 2013; Tröger et al., 2015] and its incorporation into handheld devices [Tsaloglou et al., 2018]) and field‐based DNA sequencing (Parker et al., 2017), making it even easier for smaller efforts to have large in‐country impacts on biodiversity science.…”

“…Just as the need for locally or institutionally based sequencers has decreased or disappeared, it is likely that, in the future, more general needs for laboratory infrastructure will continue to decrease. Advances are being made in all relevant areas, including DNA amplification (e.g., isothermal PCR [Boyle et al, 2013;Tröger et al, 2015] and its incorporation into handheld devices [Tsaloglou et al, 2018]) and field-based DNA sequencing (Parker et al, 2017), making it even easier for smaller efforts to have large in-country impacts on biodiversity science.…”