RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing

Abstract

Despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use Illumina to sequence small (~400 bp) hypervariable regions of the 16S rRNA SSU for phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biases of sequencing small portions can be removed, allowing for more accurate classification with deeper taxonomic resolution. With Nanopore sequencing now providing raw simplex reads with quality scores above Q20 using the kit 12 chemistry, the ease, cost, and portability of Nanopore play a leading role in performing differential bacterial abundance analysis. Sequencing the near-entire rrn operon of bacteria and archaea enables the use of the universally conserved operon holding evolutionary polymorphisms for taxonomic resolution. Here, a reproducible and validated pipeline was developed, RRN-operon Enabled Species-level Classification Using EMU (RESCUE), to facilitate the sequencing of bacterial rrn operons and to support import into phyloseq. Benchmarking RESCUE showed that fully processed reads are now parallel or exceed the quality of Sanger, with median quality scores of approximately Q20+, using the R10.4 and Guppy SUP basecalling. The pipeline was validated through two complex mock samples, the use of multiple sample types, with actual Illumina data, and across four databases. RESCUE sequencing is shown to drastically improve classification to the species level for most taxa and resolves erroneous taxa caused by using short reads such as Illumina.

Nanopore duplex sequencing as an alternative to Illumina MiSeq sequencing for eDNA-based biomonitoring of coastal aquaculture impacts

“…Taxonomic classification of Illumina-derived ASVs and Nanopore-derived OTUs was conducted using the 16S BugSeq pipeline (Fan et al 2021;Jung and Chorlton 2021). As the results of taxonomic classifications can be notably influenced by the methodology of assignment and by the reference database (Petrone et al 2023;Zhang et al 2023), we chose the same classification pipeline for both the Illumina ASVs and the Nanopore OTUs. BugSeq's 16S pipeline employs the QIIME2 VSEARCH consensus classifier (Rognes et al 2016;Hall and Beiko 2018).…”

“…Older et al (2023) found an average of 59% of 16S rRNA gene sequence reads that have passed a Phred20 filter. Petrone et al (2023) reported an average Phred score of 18.1 with an average of 79% of reads passing a Phred15 filter. Similarly, Zhang et al (2023) obtained an average Phred score of 18.8 for their bacterial 16S rRNA gene dataset, but did not apply any Phred quality filter for downstream analyses.…”

“…However, due to the maximum amplicon size of ca. 500 bp (MiSeq platform, Ravi et al (2018)) Illumina sequencing has a limited capacity regarding the taxonomic assignment of these short-read amplicons compared to the full-length 16S rRNA genes: in general, short-read Illumina amplicons, such as the V3-V4 16S rRNA gene, cannot be reliably assigned to lower taxonomic levels (genus and species ranks) (Older et al 2023;Petrone et al 2023;Zhang et al 2023).…”

“…A reliable identification of bacterial species, based on 16S rRNA genes, requires the (nearly) complete sequence information included in this taxonomic marker gene (Johnson et al 2019;Petrone et al 2023). Therefore, specific molecular bioindicators in current biomonitoring protocols that were inferred from short-read Illumina datasets are typically so-called ASVs (amplicon sequence variants), often without a taxonomic framework.…”

“…However, prior research employing Q20+ chemistry has already conducted a comparison between the Illumina MiSeq platform and ONT sequencing. It revealed that the longer sequencing lengths can offset diminished sequence quality, enabling a comparable identification of bacterial communities at higher taxonomic levels between the two platforms (Older et al 2023;Petrone et al 2023;Zhang et al 2023). Most of these studies have analysed standardised bacterial mock communities, with limited relevance for biomonitoring and aquaculture (Older et al 2023).…”