Biology subjects

Srivathsan, A.

Publications and source records attributed to Srivathsan, A..

23 records · Page 2Linked to original sources

bioRxiv2019

Faecal DNA to the rescue: Shotgun sequencing of non-invasive samples reveals two subspecies of Southeast Asian primates to be Critically Endangered species

A significant number of Southeast Asian mammal species described in the 19th and 20th century were subsequently synonymized and are now considered subspecies. Many are affected by rapid habitat loss and there is thus an urgent need to re-assess the conservation status based on species boundaries established with molecular data. However, such data are lacking for many populations and subspecies. We document via a literature survey and empirical study how shotgun sequencing of faecal DNA is a still underutilized but powerful tool for accelerating such evaluations. We obtain 11 mitochondrial genomes for three subspecies in the langur genus Presbytis through shotgun sequencing of faecal DNA (P. femoralis femoralis, P. f. percura, P. siamensis cf. cana). The genomes support the resurrection of all three subspecies to species based on multiple species delimitation algorithms (PTP, ABGD, Objective Clustering) applied to a dataset covering 40 species and 43 subspecies of Asian colobines. For two of the newly recognized species (P. femoralis, P. percura), the results lead to an immediate change in the IUCN status to Critically Endangered due to small population estimates and fragmented habitat. We conclude that faecal DNA should be more widely used for clarifying species boundaries in endangered mammals.

zoology↗

bioRxiv2019

MinION sequencing of seafood in Singapore reveals creatively labelled flatfishes, confused roe, pig DNA in squid balls, and phantom crustaceans

Food mislabelling is a growing world-wide problem that is increasingly addressed through the authentication of ingredients via techniques like mass spectrometry or DNA-sequencing. However, traditional DNA sequencing methods are slow, expensive, and require well-equipped laboratories. We here test whether these problems can be overcome through the use of Nanopore sequencing. We sequenced 92 single and 13 mixed-species samples bought in supermarkets and restaurants in Singapore which has a large and diverse seafood trade. We successfully obtained DNA barcodes for 94% and 100% of the single- and mixed-species products after correcting the numerous sequencing errors of MinION reads with a correction pipeline optimized for DNA barcodes. We find comparatively low levels of clear-cut mislabelling for single-species samples (7.6 %) while the rates are higher for mixed-species samples (38.5 %). These low rates are somewhat deceptive, however, because of the widespread use of vague common species names that do not allow for a precise assessment of the expected ingredients. With regard to the clearly mislabelled single-species products, higher-value products (e.g., prawn roe, wild-caught Atlantic salmon, halibut) are replaced with lower-value ingredients (e.g., fish roe, Pacific salmon, arrowtooth flounder) while more serious problems are observed for mixed-species samples. Cuttlefish and prawn balls repeatedly contained pig DNA and 100% of all mixed samples labelled as containing crustaceans ( crab, prawn, lobster) only yielded fish barcodes. We conclude that there is a need for more regular testing of seafood samples and suggest that due to speed and low-cost, MinION would be a good instrument for this purpose. We also emphasize the need for developing clearer labelling guidelines.

genomics↗

bioRxiv2019

1D MinION sequencing for large-scale species discovery: 7000 scuttle flies (Diptera: Phoridae) from one site in Kibale National Park (Uganda) revealed to belong to >650 species

BackgroundMore than 80% of all animal species remain unknown to science. Most of these species live in the tropics and belong to animal taxa that combine small body size with high specimen abundance and large species richness. For such clades, using morphology for species discovery is slow because large numbers of specimens must be sorted using detailed microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA sequences could be used for sorting specimens to species. Morphological verification of such \"molecular Operational Taxonomic Units\" (mOTUs) could then be based on dissection of a small subset of specimens. However, this approach requires cost-effective and low-tech DNA barcoding techniques because well equipped, well-funded molecular laboratories are not readily available in many biodiverse countries.\n\nResultsWe here document how MinION sequencing can be used for large-scale species discovery in a specimen- and species-rich taxon like the hyper-diverse fly family Phoridae (Diptera). We sequenced 7,059 specimens collected in a single Malaise trap in Kibale National Park, Uganda over the short period of eight weeks. We discovered >650 species which exceeded the number of phorid species currently described for the entire Afrotropical region. The barcodes were obtained using an improved low-cost MinION pipeline that increased the barcoding capacity sevenfold from 500 to 3,500 barcodes per flowcell. This was achieved by adopting 1D sequencing, re-sequencing weak amplicons on a used flowcell, and improving demultiplexing. Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% accuracy, 0.46% Ns) and thus yielded very similar species units (match ratio: 0.991). Morphological examination of 100 mOTUs also confirmed good congruence with morphology (93% of mOTUs; >99% of specimens) and revealed that 90% of the putative species belong to a neglected, megadiverse genus (Megaselia). We demonstrate for one Megaselia species how the molecular data can guide the description of a new species (Megaselia sepsioides sp. nov.).\n\nConclusionsWe document that one field site in Africa can be home to an estimated 1000 species of phorids and speculate that the Afrotropical diversity could exceed 100,000 species. We furthermore conclude that low-cost MinION sequencers are very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. MinION sequencing could quickly reveal the extent of the unknown diversity and is especially suitable for biodiverse countries with limited access to capital-intensive sequencing facilities.

evolutionary biology↗

bioRxiv2019

Mini-barcodes are more suitable for large-scale species discovery in Metazoa than full-length barcodes

New techniques for the species-level sorting of millions of specimens are needed in order to accelerate species discovery, determine how many species live on earth, and develop efficient biomonitoring techniques. These sorting methods should be reliable, scalable and cost-effective, as well as being largely insensitive to low-quality genomic DNA, given that this is usually all that can be obtained from museum specimens. Mini-barcodes seem to satisfy these criteria, but it is unclear how well they perform for species-level sorting when compared to full-length barcodes. This is here tested based on 20 empirical datasets covering ca. 30,000 specimens and 5,500 species, as well as six clade-specific datasets from GenBank covering ca. 98,000 specimens for over 20,000 species. All specimens in these datasets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (3 windows: 100-bp, 200-bp, 300-bp) and by excising nine mini-barcodes with established primers (length: 94 - 407-bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular Operational Taxonomic Units (mOTUs) that were obtained using three different species delimitation techniques (PTP, ABGD, objective clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length (>200-bp). Only very short mini-barcodes (<200-bp) perform poorly, especially when they are located near the 5 end of the Folmer region. The mean congruence between morphospecies and mOTUs is ca. 75% for barcodes >200-bp and the congruent mOTUs contain ca. 75% of all specimens. Most conflict is caused by ca. 10% of the specimens that can be identified and should be targeted for re-examination in order to efficiently resolve conflict. Our study suggests that large-scale species discovery, identification, and metabarcoding can utilize mini-barcodes without any demonstrable loss of information compared to full-length barcodes.

evolutionary biology↗

bioRxiv2018

A MinION-based pipeline for fast and cost-effective DNA barcoding

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory, is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION and demonstrate that one flowcell can generate barcodes for [~]500 specimens despite high base-call error rates of MinION. The pipeline overcomes the errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. These barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. We thus complement the barcode caller with an optional error correction pipeline that uses conserved amino-acid motifs from publicly available barcodes to correct the indel errors. 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 (1) 511 specimens of a mixed Diptera sample, (2) 575 specimens of ants, and (3) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION barcodes for 490 specimens which were assessed against reference Sanger barcodes (N=471). Overall, the MinION barcodes have an accuracy of 99.3%-100% and the number of ambiguities ranges from <0.01-1.5% depending on which correction pipeline is used. We demonstrate that it requires only 2 hours of sequencing to gather all information that is needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1000 barcodes can be generated in one flowcell and that the cost of a MinION barcode can be <USD 2.

bioinformatics↗