Sequencing and PCR errors are a major challenge when characterizing genetic diversity using high-throughput amplicon sequencing (HTAS). We have developed a multiplexed HTAS method, MAUI-seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. Erroneous sequences are recognized because, across the data set, they are over-represented among the minor sequences associated with UMIs. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming dada2 and unoise3, and the ability to confidently recognize genuine alleles that are present at low abundance or resemble chimeras. The method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.
Keywords: amplicon sequence variant; chimeric amplicons; error correction; high-throughput amplicon sequencing; metabarcoding.
© 2020 John Wiley & Sons Ltd.