Genomic prediction using low-coverage portable Nanopore sequencing.

Most traits in livestock, crops and humans are polygenic, that is, a large number of loci contribute to genetic variation. Effects at these loci lie along a continuum ranging from common low-effect to rare high-effect variants that cumulatively contribute to the overall phenotype. Statistical methods to calculate the effect of these loci have been developed and can be used to predict phenotypes in new individuals. In agriculture, these methods are used to select superior individuals using genomic breeding values; in humans these methods are used to quantitatively measure an individual's disease risk, termed polygenic risk scores. Both fields typically use SNP array genotypes for the analysis. Recently, genotyping-by-sequencing has become popular, due to lower cost and greater genome coverage (including structural variants). Oxford Nanopore Technologies' (ONT) portable sequencers have the potential to combine the benefits genotyping-by-sequencing with portability and decreased turn-around time. This introduces the potential for in-house clinical genetic disease risk screening in humans or calculating genomic breeding values on-farm in agriculture. Here we demonstrate the potential of the later by calculating genomic breeding values for four traits in cattle using low-coverage ONT sequence data and comparing these breeding values to breeding values calculated from SNP arrays. At sequencing coverages between 2X and 4X the correlation between ONT breeding values and SNP array-based breeding values was > 0.92 when imputation was used and > 0.88 when no imputation was used. With an average sequencing coverage of 0.5x the correlation between the two methods was between 0.85 and 0.92 using imputation, depending on the trait. This suggests that ONT sequencing has potential for in clinic or on-farm genomic prediction, however, further work to validate these findings in a larger population still remains.

Detection of Circulating RNA Using Nanopore Sequencing.

RNA sequencing using nanopore sequencing is a powerful method for transcriptome analysis. The approach is appropriate for comprehensive profiling of the wide range of long noncoding RNAs. Use of nanopore-based sequencing can provide information on novel transcripts, sequence polymorphisms, and splicing variants, and thus has advantages over other gene expression profiling methods such as microarrays. Circulating extracellular long noncoding RNAs are of particular interest because of their potential use as biomarkers. Here, we describe a protocol for cDNA-PCR sequencing of circulating RNA for biomarker discovery in whole blood samples using commercially available kits and nanopore sequencing.

Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution.

BACKGROUND: Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. RESULTS: We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. CONCLUSIONS: Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

Single-molecule nanopore sequencing reveals extreme target copy number heterogeneity in arylomycin-resistant mutants.

Tandem gene amplification is a frequent and dynamic source of antibiotic resistance in bacteria. Ongoing expansions and contractions of repeat arrays during population growth are expected to manifest as cell-to-cell differences in copy number (CN). As a result, a clonal bacterial culture could comprise subpopulations of cells with different levels of antibiotic sensitivity that result from variable gene dosage. Despite the high potential for misclassification of heterogenous cell populations as either antibiotic-susceptible or fully resistant in clinical settings, and the concomitant risk of inappropriate treatment, CN distribution among cells has defied analysis. Here, we use the MinION single-molecule nanopore sequencer to uncover CN heterogeneity in clonal populations of Escherichia coli and Acinetobacter baumannii grown from single cells isolated while selecting for resistance to an optimized arylomycin, a member of a recently discovered class of Gram-negative antibiotic. We found that gene amplification of the arylomycin target, bacterial type I signal peptidase LepB, is a mechanism of unstable arylomycin resistance and demonstrate in E. coli that amplification instability is independent of RecA. This instability drives the emergence of a nonuniform distribution of lepB CN among cells with a range of 1 to at least 50 copies of lepB identified in a single clonal population. In sum, this remarkable heterogeneity, and the evolutionary plasticity it fuels, illustrates how gene amplification can enable bacterial populations to respond rapidly to novel antibiotics. This study establishes a rationale for further nanopore-sequencing studies of heterogeneous cell populations to uncover CN variability at single-molecule resolution.

Readfish enables targeted nanopore sequencing of gigabase-sized genomes.

Nanopore sequencers can be used to selectively sequence certain DNA molecules in a pool by reversing the voltage across individual nanopores to reject specific sequences, enabling enrichment and depletion to address biological questions. Previously, we achieved this using dynamic time warping to map the signal to a reference genome, but the method required substantial computational resources and did not scale to gigabase-sized references. Here we overcome this limitation by using graphical processing unit (GPU) base-calling. We show enrichment of specific chromosomes from the human genome and of low-abundance organisms in mixed populations without a priori knowledge of sample composition. Finally, we enrich targeted panels comprising 25,600 exons from 10,000 human genes and 717 genes implicated in cancer, identifying PML-RARA fusions in the NB4 cell line in <15 h sequencing. These methods can be used to efficiently screen any target panel of genes without specialized sample preparation using any computer and a suitable GPU. Our toolkit, readfish, is available at https://www.github.com/looselab/readfish .

Benchmarking the MinION: Evaluating long reads for microbial profiling.

Nanopore based DNA-sequencing delivers long reads, thereby simplifying the decipherment of bacterial communities. Since its commercial appearance, this technology has been assigned several attributes, such as its error proneness, comparatively low cost, ease-of-use, and, most notably, aforementioned long reads. The technology as a whole is under continued development. As such, benchmarks are required to conceive, test and improve analysis protocols, including those related to the understanding of the composition of microbial communities. Here we present a dataset composed of twelve different prokaryotic species split into four samples differing by nucleic acid quantification technique to assess the specificity and sensitivity of the MinION nanopore sequencer in a blind study design. Taxonomic classification was performed by standard taxonomic sequence classification tools, namely Kraken, Kraken2 and Centrifuge directly on reads. This allowed taxonomic assignments of up to 99.27% on genus level and 92.78% on species level, enabling true-positive classification of strains down to 25,000 genomes per sample. Full genomic coverage is achieved for strains abundant as low as 250,000 genomes per sample under our experimental settings. In summary, we present an evaluation of nanopore sequence processing analysis with respect to microbial community composition. It provides an open protocol and the data may serve as basis for the development and benchmarking of future data processing pipelines.

Evaluation of a portable nanopore-based sequencer for detection of viruses in water.

The newly emerged nanopore sequencing technology such as MinION™ allows for real-time detection of long DNA/RNA fragments on a portable device, yet few have examined its performance for environmental viromes. Here we seeded one RNA virus bacteriophage MS2 and one DNA virus bacteriophage PhiX174 into 10 L well water at three levels ranging from 1 to 21,100 plaque-forming units (PFU)/mL. Two workflows were established to maximize the number of sequencing reads of RNA and DNA viruses using MinION™. With dead-end ultrafiltration, PEG precipitation, and random amplification, MinION™ was capable of detecting MS2 at 155 PFU/mL and PhiX174 at 1-2 PFU/mL. While the DNA workflow only detected PhiX174, the RNA workflow detected both MS2 and PhiX174. The virus concentration, or relative abundance of viral nucleic acids in total nucleic acids, is critical to the proportion of viral reads in sequencing results. Our findings also highlight the importance of including control samples in sequencing runs for environmental water samples with low virus abundance.

Recent advances in the detection of base modifications using the Nanopore sequencer.

DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.

Human short tandem repeat identification using a nanopore-based DNA sequencer: a pilot study.

Short tandem repeats (STRs) are repetitive DNA sequences that are highly polymorphic and widely used for personal identification in the field of forensic medicine. The standard method for determining the repeat number of STRs is capillary electrophoresis of PCR products; however, the use of DNA sequencing has increased because it can identify same-sized alleles with nucleotide substitutions (iso-alleles). In this study, we performed human STR genotyping using a portable nanopore-based DNA sequencer, the MinION, and evaluated its performance. Because the sequence quality obtained by MinION is considerably lower than those obtained with other DNA sequencers, we developed an original scoring scheme for judging the genotypes from MinION reads. Analysis of seven human samples for 21-45 STR loci yielded an average of 857 thousand reads per sample, and the accuracy of genotyping and iso-allele identification reached 75.7% and 82%, respectively. Although the accuracy is higher than that reported previously, further improvements are required before this method can be practically applied.

Entirely Off-Grid and Solar-Powered DNA Sequencing of Microbial Communities during an Ice Cap Traverse Expedition.

Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth's surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging environmental conditions. While remote research stations have significantly lowered the barrier to studying the microbial communities on icecaps, their use has led to a bias for data collection in the near vicinity of these institutions. Here, miniaturisation of a DNA sequencing lab suitable for off-grid metagenomic studies is demonstrated. Using human power alone, this lab was transported across Europe's largest ice cap (Vatnajökull, Iceland) by ski and sledge. After 11 days of unsupported polar-style travel, a metagenomic study of a geothermal hot spring gorge was conducted on the remote northern edge of the ice cap. This tent-based metagenomic study resulted in over 24 h of Nanopore sequencing, powered by solar power alone. This study demonstrates the ability to conduct DNA sequencing in remote locations, far from civilised resources (mechanised transport, external power supply, internet connection, etc.), whilst greatly reducing the time from sample collection to data acquisition.

Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies: Current Uses and Future Directions.

We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology's MinION portable sequencing platform, as well as summarize areas of recent development.

A multiplex pharmacogenetics assay using the MinION nanopore sequencing device.

OBJECTIVES: The MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. As a proof of principle, we developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, the two commonly used drugs that show response variability due to genetic polymorphisms. METHODS: Six reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting 27 key variants. These products were then barcoded to enable sample multiplexing in one sequencing run. Four variant calling tools (marginCaller, VarScan 2, nanopolish, Clairvoyante) were used to compare genotyping accuracy. RESULTS: In our cohort, 81 out of 84 samples were successfully sequenced and genotyped. Using nanopolish as the variant calling tool achieved accuracy >95% for all except two variants. A known single base deletion (CYP2C9*6) was successfully detected. CONCLUSION: While minor misgenotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays using small PCR amplicons are possible on the MinION sequencing device.

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules.

Our vision of DNA transcription and splicing has changed dramatically with the introduction of short-read sequencing. These high-throughput sequencing technologies promised to unravel the complexity of any transcriptome. Generally gene expression levels are well-captured using these technologies, but there are still remaining caveats due to the limited read length and the fact that RNA molecules had to be reverse transcribed before sequencing. Oxford Nanopore Technologies has recently launched a portable sequencer which offers the possibility of sequencing long reads and most importantly RNA molecules. Here we generated a full mouse transcriptome from brain and liver using the Oxford Nanopore device. As a comparison, we sequenced RNA (RNA-Seq) and cDNA (cDNA-Seq) molecules using both long and short reads technologies and tested the TeloPrime preparation kit, dedicated to the enrichment of full-length transcripts. Using spike-in data, we confirmed that expression levels are efficiently captured by cDNA-Seq using short reads. More importantly, Oxford Nanopore RNA-Seq tends to be more efficient, while cDNA-Seq appears to be more biased. We further show that the cDNA library preparation of the Nanopore protocol induces read truncation for transcripts containing internal runs of T's. This bias is marked for runs of at least 15 T's, but is already detectable for runs of at least 9 T's and therefore concerns more than 20% of expressed transcripts in mouse brain and liver. Finally, we outline that bioinformatics challenges remain ahead for quantifying at the transcript level, especially when reads are not full-length. Accurate quantification of repeat-associated genes such as processed pseudogenes also remains difficult, and we show that current mapping protocols which map reads to the genome largely over-estimate their expression, at the expense of their parent gene.