MetaPlex: An Ion Torrent COI metabarcoding workflow and toolkit to
increase experimental efficacy and efficiency
Abstract
DNA barcoding has become a dependable way to assign taxonomic
identifications to otherwise unknown DNA samples. Metabarcoding using
next-generation sequencing (NGS) maintains this same caliber of
taxonomic identification and can occur on a mass scale in pooled, or
multiplexed, samples from a wide variety of sources when properly
multiplexed. However, this methodology has several pitfalls, including
inaccurate source sample assignment, multi-step workflows that leave
room for human error, and high costs. To combat these issues, library
preparation protocols have been established for the popular Illumina
sequencing platform which reduce sequence assignment errors to levels
equivalent to background noise, while also greatly reducing cost. An
equivalent protocol has yet to be established for the Ion Torrent
sequencing ecosystem. Here, we present MetaPlex, a library preparation
workflow and post-processing toolkit for efficient and accurate COI
metabarcoding on Ion Torrent sequencers. These methods significantly
decrease the costs of multiplexed sample sequencing by nearly 9x when
compared to commercially available kits through the reduction of
reagents needed for library preparation. In addition, our workflow
provides increased reliability through elimination of laboratory
processes which are known to negatively bias NGS outputs and
source-sample tracing, limiting the common pitfall known as index jumps
to 0.17%. Finally, our accompanying bioinformatic toolkit increases
user accessibility by providing an easy-to-use command line tool for
processing and correcting for errors in our, and others’, dual-indexed
reads.