Abstract
BackgroundNanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics. However, several applications are limited by relatively high single-read error rates (>10 %), including RNA-seq, haplotype sequencing and 16S sequencing.ResultsWe developed the Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) as a strategy for obtaining long and accurate nanopore reads, starting with low input DNA. Applying INC-Seq for 16S rRNA-based bacterial profiling generated full-length amplicon sequences with a median accuracy >97 %.ConclusionsINC-Seq reads enabled accurate species-level classification, identification of species at 0.1 % abundance and robust quantification of relative abundances, providing a cheap and effective approach for pathogen detection and microbiome profiling on the MinION system.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-016-0140-7) contains supplementary material, which is available to authorized users.
Highlights
Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics
The MinION sequencing platform introduced by Oxford Nanopore Technologies (ONT) is a portable and inexpensive device that generates long reads in real time and can further democratize genomics [1, 2]
Pacific Biosciences (PacBio) circular consensus sequencing (CCS) [8] ( known as single molecule, real-time (SMRT) CCS) reads through circularized templates repeatedly by synthesis but cannot be directly applied to nanopore sequencing that requires a single stranded linear template to be threaded through the nanopore channel
Summary
Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics. Raw single-strand reads (1D reads) on the MinION platform have been reported to have an error rate >20 %, but more accurate reads (10-15 % median error rate) are typically generated by a protocol that reads the complementary strand and computes an in silico consensus (2D reads) [6]. Similar to this idea, experimental protocols have been developed on other sequencing platforms to boost read accuracy by repeatedly sequencing the same parent template molecule, which generates more reliable consensus reads [7,8,9,10]. Circ-Seq [9, 10] generates linear templates (3 repeating units on average) with a
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