NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform

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.

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

“…PacBio HiFi sequencing mode produces highly accurate reads using their circular consensus sequencing (CCS) technology, reducing raw read error to < 1% (Wenger et al 2019). Similarly, rolling circle amplification can be used in metabarcoding applications to mitigate error rates of the nanopore-based sequencing platforms (Calus et al 2018).…”

Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations

“…Our HTS data are based on the pyrosequencing 454 method (Roche) that has been replaced by newer platforms that provide sequencing depth orders of magnitude higher, such as Illumina or Oxford Nanopore. However, as the main biases arise from DNA extraction, PCR amplification, and uneven 16S and 18S rRNA gene copy numbers (47), these newer methods will not necessarily improve the quantitative accuracy of the sequencing data, as shown with mock communities sequenced using Illumina (13)(14)(15) and Oxford Nanopore (48,49) platforms. In contrast, the relative abundances of sequences obtained from the same samples by both pyrosequencing and Illumina correlated very strongly (r 2 Ͼ 0.99) (50).…”

Nanopore sequencing of microbial communities reveals the potential role of sea lice as a reservoir for fish pathogens

“…This technology allows the sequencing of the full-16S rRNA gene, increasing the capacity for taxonomical assignation, and it can be performed in real-time with reduced equipment, increasing its potential as a field tool for remote locations 23 . However, despite these advantages, when comparing to short reads sequencing such as Illumina, the error rate is higher 22 , problem that has been progressively mitigated with technological advances and better bioinformatic tools 24,25 . Regarding the study of microbiota, several studies have been dedicated to compare taxonomical analysis derived by this technology with Illumina sequencing, and have shown that error rate has been compensated with larger fragments, providing in some cases higher taxonomical resolution 26,27 .…”