TY - JOUR T1 - 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 JF - bioRxiv DO - 10.1101/622365 SP - 622365 AU - Amrita Srivathsan AU - Emily Hartop AU - Jayanthi Puniamoorthy AU - Wan Ting Lee AU - Sujatha Narayanan Kutty AU - Olavi Kurina AU - Rudolf Meier Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/04/29/622365.abstract N2 - Background Most animal species remain to be discovered. We recently proposed to tackle this problem using a ‘reverse workflow’ where all specimens are barcoded via tagged amplicon sequencing and then sorted into putative species (mOTUs). We furthermore suggested that the COI barcodes can be obtained with minimal laboratory equipment using MinION sequencing, but our test with 1D2 reads only yielded 500 barcodes per flowcell.Results Here we show how MinION 1D sequencing can be used to obtain ~3500 COI barcodes per flowcell. Based on 7062 MinION barcodes for a hyper-diverse family of flies (Diptera: Phoridae) collected by one trap in Kibale National Park, Uganda, we discover ~650 species which exceeds the number of phorid species described for the entire Afrotropical region. Our updated MinION pipeline increases processing speed via parallelization, improves demultiplexing, and yet yields reliable barcodes (99.99% accuracy) and similar mOTUs as Illumina sequencing (match ratio: 0.989). Morphological examination of 100 mOTUs confirms good congruence (93% of mOTUs; >99% of specimens). Nearly 90% of species and specimens belong to the megadiverse genus Megaselia which is routinely neglected because its species diversity and abundance is too overwhelming. We show that it can be tackled with the reverse workflow. We also illustrate how the molecular data guides the description of a new species: Megaselia sepsioides sp. nov..Conclusions MinION is suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. Approximately, 3500 specimens can be sequenced using one MinION flowcell at a barcode cost of <0.35 USD. ER -