DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory and is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION™ reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION™ barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N = 471). Overall, the MinION™ barcodes have an accuracy of 99.3%-100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be

Freshwater habitats are of high conservation value and provide a wide range of ecosystem services. Effective management requires regular monitoring. However, conventional methods based on direct observation or specimen collection are so invasive, expensive and labour-intensive that frequent monitoring is uncommon. Here, we test whether the evaluation of environmental DNA (eDNA) from water based on a simple protocol can be used for assessing biodiversity. We use universal metazoan primers for characterizing water eDNA across horizontal and vertical spatial dimensions in two reservoirs with known species diversity for two key taxa. eDNA obtained directly from 42 samples × 15 ml water (total = 630 ml) per reservoir yielded DNA signatures for more than 500 metazoan species, of which 105 could be identified to species/genus based on DNA barcodes. We show that eDNA can be used to assign each water sample to its reservoir of origin, and that eDNA outperforms conventional survey methods in single-sample richness comparisons, while revealing evidence for hundreds of unknown species that are undetected by conventional bioassessment methods. eDNA also confirms the presence of a recently discovered invasive snail species and provides evidence for the continued survival of a rare native species of goby not sighted in that habitat since 2007. eDNA thus promises to be a useful addition to the bioassessment toolbox for freshwater systems.

20DNA barcodes are useful for species discovery and species identification, but obtaining 21 barcodes currently requires a well-equipped molecular laboratory, is time-consuming, and/or 22 expensive. We here address these issues by developing a barcoding pipeline for Oxford 23 Nanopore MinION™ and demonstrate that one flowcell can generate barcodes for ~500 24 specimens despite high base-call error rates of MinION™. The pipeline overcomes the 25 errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. 26These barcodes are overall mismatch-free but retain indel errors that are concentrated in 27 homopolymeric regions. We thus complement the barcode caller with an optional error 28 correction pipeline that uses conserved amino-acid motifs from publicly available barcodes 29 to correct the indel errors. The effectiveness of this pipeline is documented by analysing 30 reads from three MinION™ runs that represent three different stages of MinION™ 31 development. They generated data for (1) 511 specimens of a mixed Diptera sample, (2) 32 575 specimens of ants, and (3) 50 specimens of Chironomidae. The run based on the latest 33 chemistry yielded MinION barcodes for 490 specimens which were assessed against 34 reference Sanger barcodes (N=471). Overall, the MinION barcodes have an accuracy of 35 99.3%-100% and the number of ambiguities ranges from <0.01-1.5% depending on which 36 correction pipeline is used. We demonstrate that it requires only 2 hours of sequencing to 37 gather all information that is needed for obtaining reliable barcodes for most specimens 38

BackgroundMacroinvertebrates such as non-biting midges (Chironomidae: Diptera) are important components of freshwater ecosystems. However, they are often neglected in biodiversity and conservation research because invertebrate species richness is difficult and expensive to quantify with traditional methods. We here demonstrate that Next Generation Sequencing barcodes (“NGS barcodes”) can provide relief because they allow for fast and large-scale species-level sorting of large samples at low cost.ResultsWe used NGS barcoding to investigate the midge fauna of Singapore’s swamp forest remnant (Nee Soon Swamp Forest). Based on > 14.000 barcoded specimens, we find that the swamp forest maintains an exceptionally rich fauna composed of an observed number of 289 species (estimated 336 species) in a very small area (90 ha). We furthermore barcoded the chironomids from three surrounding reservoirs that are located in close proximity. Although the swamp forest remnant is much smaller than the combined size of the freshwater reservoirs in the study (90 ha vs. > 450 ha), the latter only contains 33 (estimated 61) species. We show that the resistance of the swamp forest species assemblage is high because only 8 of the 314 species are shared despite the close proximity. Moreover, shared species are not very abundant (3% of all specimens). A redundancy analysis revealed that ~ 21% of the compositional variance of midge communities within the swamp forest was explained by a range of variables with conductivity, stream order, stream width, temperature, latitude (flow direction), and year being significant factors influencing community structure. An LME analysis demonstrates that the total species richness decreased with increasing conductivity.ConclusionOur study demonstrates that midge diversity of a swamp forest can be so high that it questions global species diversity estimates for Chironomidae, which are an important component of many freshwater ecosystems. We furthermore demonstrate that small and natural habitat remnants can have high species turnover and can be very resistant to the invasion of species from neighboring reservoirs. Lastly, the study shows how NGS barcodes can be used to integrate specimen- and species-rich invertebrate taxa in biodiversity and conservation research.Electronic supplementary materialThe online version of this article (10.1186/s12983-018-0276-7) contains supplementary material, which is available to authorized users.

Metabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short‐read high‐throughput sequencing platforms. The Oxford Nanopore MinIONTM, a portable sequencing platform, represents a low‐cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%. To test whether the MinIONTM represents a viable alternative to other sequencing platforms, we used rolling circle amplification (RCA) to generate full‐length consensus DNA barcodes for a bulk mock sample of 50 aquatic invertebrate species with at least 15% genetic distance to each other. By applying two different laboratory protocols, we generated two MinIONTM runs that were used to build error‐corrected consensus sequences. A newly developed Python pipeline, ASHURE, was used for data processing, consensus building, clustering and taxonomic assignment of the resulting reads. Our pipeline achieved median accuracies of up to 99.3% for long concatemeric reads (>45 barcodes) and successfully identified all 50 species in the mock community. The use of RCA was integral for increasing consensus accuracy but was also the most time‐consuming step of the laboratory workflow. Most concatemeric reads were skewed towards a shorter read length range with a median read length of up to 1,262 bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding, but exploration of other isothermal amplification procedures to improve consensus accuracy is recommended.

18Metabarcoding has become a common approach to the rapid identification of the species 19 composition in a mixed sample. The majority of studies use established short-read high-throughput 20 sequencing platforms. The Oxford Nanopore MinION TM , a portable sequencing platform, represents a 21 low-cost alternative allowing researchers to generate sequence data in the field. However, a major 22 be used for metabarcoding but we recommend the exploration of other isothermal amplification 36 procedures to improve consensus length. 37 38 39 452We thank all staff at the CBG who helped to collect the samples employed to assemble the mock 453 community. We also would like to thank Florian Leese, Arne Beermann, Cristina Hartmann-Fatu, and 454Marie Gutgesell for collecting and providing specimens.

State-of-the-art nanopore sequencing enables rapid and real-time identification of novel pathogens, which has wide application in various research areas and is an emerging diagnostic tool for infectious diseases including COVID-19. Nanopore translocation enables de novo sequencing with long reads (> 10 kb) of novel genomes, which has advantages over existing short-read sequencing technologies. Biological nanopore sequencing has already achieved success as a technology platform but it is sensitive to empirical factors such as pH and temperature. Alternatively, ångström- and nano-scale solid-state nanopores, especially those based on two-dimensional (2D) membranes, are promising next-generation technologies as they can surpass biological nanopores in the variety of membrane materials, ease of defining pore morphology, higher nucleotide detection sensitivity, and facilitation of novel and hybrid sequencing modalities. Since the discovery of graphene, atomically-thin 2D materials have shown immense potential for the fabrication of nanopores with well-defined geometry, rendering them viable candidates for nanopore sequencing membranes. Here, we review recent progress and future development trends of 2D materials and their ångström- and nano-scale pore-based nucleic acid (NA) sequencing including fabrication techniques and current and emerging sequencing modalities. In addition, we discuss the current challenges of translocation-based nanopore sequencing and provide an outlook on promising future research directions.

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