Portable Nanopore Sequencing Research Articles

Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer.

The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems.

Portable Nanopore Sequencing for Viral Surveillance.

The recent Nature article (1) entitled, “Real-Time, Portable Genome Sequencing for Ebola Surveillance,” reports nanopore sequencing of the Ebola virus from 142 subjects near the end of the outbreak in West Africa from March to October 2015. The nanopore sequencer (MinION, Oxford Nanopore Technologies) has a mass of 87 g and plugs into a laptop computer by way of a USB 3.0 interface and cable. The consumable flow cell has up to 512 channels available for sequencing, each of which can simultaneously read single strands of DNA and their ligated complementary strands as “2D” reads, providing correlated data on both strands of DNA. The nanopores are typically made up of proteins like α-hemolysin that are pore-forming bacterial toxins embedded in an electrically insulating membrane. A voltage between the 2 sides of the membrane attracts DNA to the pore in an electrolyte solution. The DNA is captured by a polymerase near the pore that paces the rate of transport to 30–280 bases/s. The passage of single-stranded DNA affects the pore current, generating electrical “squiggle signals” that are sequence-specific. In simple terms, this is the Coulter principle applied to molecules rather than cells. In half of the cases, sufficient reads (25 2D reads for each amplicon) were obtained in <1 h of instrument time with a mean passing rate of 36.3%. The mean read rate was 138 amplicons/min, and in some cases, only 15 min of sequencing was required. Custom offline acquisition software was provided by the company so that Internet communication was not initially required. Nanopore sequencing is conceptually elegant and data acquisition is fast. However… Before sequencing, sample transport and preparation are required. To prevent delays of air transport of samples, a portable 50-kg laboratory was transported in airline luggage to Guinea. As samples became available locally, they were transported …

Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer.

BackgroundThe miniaturised and portable DNA sequencer MinION™ has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinION™ system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences.ResultsUsing R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90 % of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using ‘universal’ primers.ConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinION™ DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.

Poretools: a toolkit for analyzing nanopore sequence data.

Motivation: Nanopore sequencing may be the next disruptive technology in genomics, owing to its ability to detect single DNA molecules without prior amplification, lack of reliance on expensive optical components, and the ability to sequence long fragments. The MinION™ from Oxford Nanopore Technologies (ONT) is the first nanopore sequencer to be commercialized and is now available to early-access users. The MinION™ is a USB-connected, portable nanopore sequencer that permits real-time analysis of streaming event data. Currently, the research community lacks a standardized toolkit for the analysis of nanopore datasets.Results: We introduce poretools, a flexible toolkit for exploring datasets generated by nanopore sequencing devices from MinION™ for the purposes of quality control and downstream analysis. Poretools operates directly on the native FAST5 (an application of the HDF5 standard) file format produced by ONT and provides a wealth of format conversion utilities and data exploration and visualization tools.Availability and implementation: Poretools is an open-source software and is written in Python as both a suite of command line utilities and a Python application programming interface. Source code is freely available in Github at https://www.github.com/arq5x/poretoolsContact: n.j.loman@bham.ac.uk and aaronquinlan@gmail.comSupplementary information: An IPython notebook demonstrating the functionality of poretools is in Github. Complete documentation is available at http://poretools.readthedocs.org.