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Decoding long nanopore sequencing reads of natural DNA.

ABSTRACT: Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

SUBMITTER: Laszlo AH

PROVIDER: S-EPMC4126851 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Decoding long nanopore sequencing reads of natural DNA.

Laszlo Andrew H AH Derrington Ian M IM Ross Brian C BC Brinkerhoff Henry H Adey Andrew A Nova Ian C IC Craig Jonathan M JM Langford Kyle W KW Samson Jenny Mae JM Daza Riza R Doering Kenji K Shendure Jay J Gundlach Jens H JH

Nature biotechnology 20140625 8

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleo ...[more]

PMID: 24964173

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Project description:BackgroundLong-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.ResultsThe assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.ConclusionsWe described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Project description:Long-read nanopore sequencing is capable of continuously reading thousands of base pairs. Here, we report the application of nanopore sequencing technology for analysing hypoxia-specific transcriptomes in normal liver and liver cancer cell lines, which help us to better understand the mechanism of cell survival and metastasis under hypoxia. We conducted transcriptomic sequencing of normal liver and liver cancer cell lines under normoxia or hypoxia, founding that long-read sequencing could detect thousands of hypoxia-specific isoforms and unannotated novel genes. At the cellular level, we have verified that the novel gene 644 is most up-regulated novel gene under hypoxic conditions. We observed that the novel gene 644 possesses classical mRNA characteristics, with its CDS region encoding 91 amino acids. Through protein structure predictions, we discovered that the novel gene 644 contains a transmembrane domain. Experimental validation confirmed that the novel gene 644 is a membrane protein, and knockdown of the novel gene 644 significantly suppresses the proliferative capacity of liver cancer cells under hypoxic conditions. Furthermore, we revealed the protein structure alteration of hypoxia specific isoforms with molecular dynamics simulation. The results indicate that our hypoxic dataset can serve as a foundation for the structural simulation of hypoxic-specific proteins. In summary, we have presented a hypoxic-specific third-generation sequencing dataset for further analysis, which provides sufficient details on cellular transcriptomes and is particularly valuable for understanding the survival and metastasis of tumor under hypoxic conditions. From this dataset, we have validated the importance of novel gene 644 for tumor survival under hypoxia and identified alterations in hypoxic-specific protein structures. These discoveries can bring us new knowledge about hypoxia, leading to the identification of potential tumor treatment targets and biomarkers.

Dataset Information

Decoding long nanopore sequencing reads of natural DNA.

Publications

Decoding long nanopore sequencing reads of natural DNA.

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