A workflow for accurate metabarcoding using nanopore MinION sequencing

Abstract

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.

Identification of plastic-associated species in the Mediterranean Sea using DNA metabarcoding with Nanopore MinION

“…Sequences were filtered based on read quality (-min_qual_mean 10) and read length was restricted (-min_len X -max_len Y as follows: 16S, 1200–1400 bp; 18S, 650–720 bp; COI, 630–700 bp; ITS, 270–340; tufA , 700–1000), using prinseq-lite V0.20.4 29 . Because generating consensus sequences is essential for increasing the accuracy in Nanopore MinION studies 30 , 31 , we generated consensus sequences for each of the amplicon read datasets using ONTrack pipeline ( https://github.com/MaestSi/ONTrack ) according to Maestri et al 32 . The consensus sequences were also used as reference sequences for the calculation of the mapping and error rates using ONTrack Calculate_mapping_rate.sh and Calculate_error_rate.sh scripts 32 .…”

Ångström- and Nano-scale Pore-Based Nucleic Acid Sequencing of Current and Emergent Pathogens

“…In addition, nanopore translocation sequencing offers easier operability as well as portability conducive for widespread use in non-traditional and resource-constrained settings. For instance, ONT's MinION device is operable in atypical laboratory settings, starting with accuracy rates of 60% and improving to more than 99% with the use of various bioinformatics pipelines [1], [13], [14].…”

“…De novo sequencing, which sequences a novel genome where a reference sequence is not available for alignment, has largely facilitated the primer design for newly emerging pathogens. Nanopore translocation technology identifies biomolecules via membrane translocation and is suitable for de novo sequencing due to its ability to sequence long reads (> 10 kb) [1]. In addition, nanopore-based sequencing has recently emerged as a promising nonfluorescence-based platform for the testing of SARS-CoV-2.…”

Takeaways from Mobile DNA Barcoding with BentoLab and MinION

“…The observed increase in samples having zero ambiguous bases points to an improved homopolymer resolution with the R10.3 chemistry. This marked improvement in R10.3 sequencing chemistry is a welcome development and paves the way for furthering nanopore sequencing applications such as DNA metabarcoding [ 82 , 83 , 84 , 85 ]. Error-prone reads from the R9 chemistry make it challenging to assign taxonomy [ 53 ], and previous studies have resorted to complex laboratory procedures [ 84 ] or reference-based polishing [ 82 ] to negate these sequencing errors.…”

“…This marked improvement in R10.3 sequencing chemistry is a welcome development and paves the way for furthering nanopore sequencing applications such as DNA metabarcoding [ 82 , 83 , 84 , 85 ]. Error-prone reads from the R9 chemistry make it challenging to assign taxonomy [ 53 ], and previous studies have resorted to complex laboratory procedures [ 84 ] or reference-based polishing [ 82 ] to negate these sequencing errors. One study tested the R10.3 chemistry for nanopore metabarcoding, but the only comparisons made to R9.4.1 chemistry were in terms of read coverage and read size distribution; no assessments were made on sequencing accuracy [ 83 ].…”