Abstract
BackgroundAmplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output.ResultsIn this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences.ConclusionsNanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.
Highlights
Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths
The DNA pool consisting of plasmid-like structures was subject to rolling circle amplification (RCA) using random hexamer-free protocol using a combination of primase/polymerase (PrimPol) and hi
The prepared amplicon pools for both single-organism and 10-organism mock community samples were subject to library preparation using the standard 2D (SQK-LSK208) and 1D2 (SQK-LSK308) kits using Oxford Nanopore Technologies (ONT) specifications and sequenced on the MinION MK1b device followed by base calling using Albacore 1.2.4
Summary
Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While excellent at bulk profiling of microbial communities through multiplexed deep sequencing, short read lengths are limited in the taxonomic resolution of sequenced reads and, more so, are not amenable to robust phylogenetic analyses to assess the relationship between sequences originating from unknown microbes with those in publicly available databases. An important effect of the proliferation in short read sequencing applications has been a decrease in the rate at which long higherquality sequences, of SSU rRNA genes, are being deposited in public databases. This effect is to some extent being
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