Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing

Abstract

1Background: 2More than 80% of all animal species remain unknown to science. Most of these species live in 3 the tropics and belong to animal taxa that combine small body size with high specimen 4abundance and large species richness. For such clades, using morphology for species 5 discovery is slow because large numbers of specimens must be sorted using detailed 6 microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA 7 sequences could be used for sorting specimens to species. Morphological verification of such 8 "molecular Operational Taxonomic Units" (mOTUs) could then be based on dissection of a small 9 subset of specimens. However, this approach requires cost-effective and low-tech DNA 10 barcoding techniques because well equipped, well-funded molecular laboratories are not readily 11 available in many biodiverse countries. 12 Results: 13We here document how MinION sequencing can be used for large-scale species discovery in a 14 specimen-and species-rich taxon like the hyper-diverse fly family Phoridae (Diptera). We 15 sequenced 7,059 specimens collected in a single Malaise trap in Kibale National Park, Uganda 16 over the short period of eight weeks. We discovered >650 species which exceeded the number 17 of phorid species currently described for the entire Afrotropical region. The barcodes were 18 obtained using an improved low-cost MinION pipeline that increased the barcoding capacity 19 sevenfold from 500 to 3,500 barcodes per flowcell. This was achieved by adopting 1D 20 sequencing, re-sequencing weak amplicons on a used flowcell, and improving demultiplexing. 21Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% 22 methods. In insects, one of the most widely used methods is Malaise trapping. Such traps 48 routinely collect thousands, or even tens of thousands, of specimens per site and week; i.e., 49 sorting all specimens to species-level virtually never happens and the world's natural history 50 museums store billions of unsorted specimens. Species-level sorting is usually restricted to a 51 few taxa with small to moderate numbers of specimens. It is accomplished in two stages. The 52 first is grouping specimens into easily identifiable major taxa (e.g., major groups of beetles, flies, 53 wasps). This type of pre-sorting is usually accomplished by parataxonomists with basic training 54 in morphology (e.g., students). The main challenge is the second sorting stage; i.e., sorting to 55 species-level. This work is best carried out by taxonomic experts whose techniques are, 56 however, mostly effective for taxa that have fairly small numbers of specimens and species. In 57 contrast, large, hyperdiverse and abundant taxa are ill-suited because they require dissection 58 and microscopic study of many specimens. An alternative to species-level sorting by 59 taxonomists is a hybrid approach that combines rapid pre-sorting to "morpho-species" by 60 parataxonomists with subsequent verification of morpho-species via DNA barcodes that ar...

MinION-based DNA barcoding of preserved and non-invasively collected wildlife samples

“…More relaxed settings reduce raw read loss, but increase the chance of including incorrectly assigned reads or reads with higher sequencing error. Srivathsan et al (2019) and Maestri et al (2019) noted similar magnitudes of read loss with ~76% and ~53.6% of reads lost after demultiplexing, respectively; other MinION DNA barcoding publications have not reported this statistic. Despite the read loss, MiniBar-and qcat-demultiplexed reads performed well based on all our metrics for accurate species identification.…”

“…The ability to use field-friendly DNA extraction protocols with these sample types will help to overcome logistical challenges, such as the need for cumbersome or expensive equipment, for wildlife field research. The accuracy of our species identifications is on par with previous MinION DNA barcoding studies and pipelines (Pomerantz et al, 2018;Srivathsan et al, 2018Srivathsan et al, , 2019Krehenwinkel, Pomerantz, Henderson, et al, 2019;Maestri et al, 2019). For all tissue types, extraction methods, and subsets tested with our pipeline, we obtained high quality reads and a consensus sequence that matched >99.29% and at least 419/421 bp to the Sanger sequence for each sample.…”

“…The Oxford Nanopore Technologies (ONT) MinION sequencer is currently the only available portable sequencer. Although nanopore sequencing is known to have higher raw sequence error rates in comparison to standard short read sequencing platforms such as Illumina or BGI-Seq, particularly at homopolymeric regions (Ip et al, 2015;Jain et al, 2017), significant improvements in the accuracy of MinION sequencing chemistry has led to its recent rise in popularity for field applications (reviewed in Srivathsan et al, 2019). This sequencer is especially useful in situations where there is a lack of access to sequencing facilities or when sample export is difficult.…”

“…To expand the potential for portable sequencing applications, field-friendly DNA extraction methods can be used to reduce lab equipment requirements. While field-friendly DNA extraction methods are often less effective at producing DNA of high concentration and purity levels, MinION DNA barcoding has been successfully performed using QuickExtract TM solution (Lucigen), which only requires a heat source (Srivathsan et al, 2019). The Chelex® 100 resin (Bio-rad) extraction method similarly only requires a heat source, but is less expensive and has not been tested for MinION sequencing.…”

“…To date, MinION DNA barcoding pipelines have used either assembly (Pomerantz et al, 2018;Krehenwinkel, Pomerantz, Henderson, et al, 2019), clustering-based (Maestri et al, 2019), or alignment (Srivathsan et al, 2018(Srivathsan et al, , 2019 methods. Assembly approaches generally work more consistently for longer barcodes (~1kb), as the underlying software were originally designed for assembling long reads for genome assemblies rather than amplicons.…”