NanoAmpli-Seq: A workflow for amplicon sequencing from mixed microbial communities on the nanopore sequencing platform
7th January 2018 - BioRxiv
Small subunit (SSU) rRNA gene sequencing on second generation platforms leverages their deep sequencing and multiplexing capacity, but is limited in genetic resolution due to short read lengths. While third generation platforms overcome this limitation, their application has been limited due to high error rates. In this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) approach, for full-length SSU rRNA gene sequencing. NanoAmpli-Seq includes key improvements to INC-Seq that reduces sample processing time while significantly improving sequence accuracy. NanoAmpli-Seq adds chopSeq for correction of INC-Seq consensus reads and nanoClust for read partitioning-based de novo clustering and within cluster consensus calling to obtain full-length 16S rRNA gene sequences. NanoAmpli-Seq accurately estimates diversity of tested mock communities with average sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymers, indicating that homopolymer aware basecalling or error correction may allow for sequence accuracy nearing 100%.