Natrix2 – Improved amplicon workflow with novel Oxford Nanopore Technologies support and enhancements in clustering, classification and taxonomic databases

By

Deep, Aman
Bludau, Dana
Welzel, Marius
Clemens, Sandra
Heider, Dominik
Boenigk, Jens
Beisser, Daniela

Type

Article

Date of Publication

2023-10-24

Original Publication

Metabarcoding and metagenomics

Volume

7

Pages

1--9

Subjects

Amplicon Sequence Variants , amplicon sequencing , community profiling , metabarcoding , microbiome , Operational Taxonomic Units , Snakemake workflow , ultra-long reads

Contributed by

Pensoft Publishers

Language

English

Published by

Bulgaria Pensoft Publishers 2017-

Identifiers

DOI: https://doi.org/10.3897/mbmg.7.109389

 

Abstract

Sequencing of amplified DNA is the first step towards the generation of Amplicon Sequence Variants (ASVs) or Operational Taxonomic Units (OTUs) for biodiversity assessment and comparative analyses of environmental communities and microbiomes. Notably, the rapid advancements in sequencing technologies have paved the way for the growing utilization of third-generation long-read approaches in recent years. These sequence data imply increasing read lengths, higher error rates, and altered sequencing chemistry. Likewise, methods for amplicon classification and reference databases have progressed, leading to the expansion of taxonomic application areas and higher classification accuracy. With Natrix, a user-friendly and reducible workflow solution, processing of prokaryotic and eukaryotic environmental Illumina sequences using 16S or 18S is possible. Here, we present an updated version of the pipeline, Natrix2, which incorporates VSEARCH as an alternative clustering method with better performance for 16S metabarcoding approaches and mothur for taxonomic classification on further databases, including PR2, UNITE and SILVA. Additionally, Natrix2 includes the handling of Nanopore reads, which entails initial error correction and refinement of reads using Medaka and Racon to subsequently determine their taxonomic classification.