Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants’ occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants’ tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants’ emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant’s tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant’s tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.

Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

Since December 2019, the emergence of a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. As consequence, a major effort worldwide has been performed for the development of viral diagnostics strategies aiming at (i) reducing the diagnostics time, (ii) decrease the costs per assay, and (iii) providing population-scale solutions. Beyond the diagnostics requirements, the description of the B.1.1.7 strain, originated in the south of England, as a highly transmissible variant has strongly accelerated the worldwide interest in tracking SARS-CoV-2 variants occurrence. Since then, other extremely infectious variants were described and unsurprisingly further others are expected to be discovered, notably due to the long period of time on which the pandemic situation is lasting. Interestingly, all currently described SARS-CoV-2 variants present systematically several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants tracking, our proposed strategy focuses on the SPIKE region, as a way to increase the number of candidate samples to screen at once; an essential aspect to accelerate SARS-CoV-2 diagnostics, but also improve variants emergence and progression surveillance. Herein we present a proof of concept study, for performing both at once, population-scale SARS-CoV-2 diagnostics and variants tracking. This strategy relies on (i) the use of the portable and affordable MinION DNA sequencer; (ii) a DNA barcoding strategy and a SPIKE gene-centered variants tracking, for largely increasing the number of candidates per assay; and (iii) a real-time diagnostics and variants tracking monitoring thanks to our software RETIVAD. As a whole, this strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.

Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants

Future of nanopore DNA sequencing: schematic illustration shows the future of nanopore DNA sequencing by using a customized biological nanopore with appropriate fabrication.

Overcoming High Nanopore Basecaller Error Rates for DNA Storage Via Basecaller-Decoder Integration and Convolutional Codes

ABSTRACTAs magnetization and semiconductor based storage technologies approach their limits, bio-molecules, such as DNA, have been identified as promising media for future storage systems, due to their high storage density (petabytes/gram) and long-term durability (thousands of years). Furthermore, nanopore DNA sequencing enables high-throughput sequencing using devices as small as a USB thumb drive and thus is ideally suited for DNA storage applications. Due to the high insertion/deletion error rates associated with basecalled nanopore reads, current approaches rely heavily on consensus among multiple reads and thus incur very high reading costs. We propose a novel approach which overcomes the high error rates in basecalled sequences by integrating a Viterbi error correction decoder with the basecaller, enabling the decoder to exploit the soft information available in the deep learning based basecaller pipeline. Using convolutional codes for error correction, we experimentally observed 3x lower reading costs than the state-of-the-art techniques at comparable writing costs.The code, data and Supplementary Material is available at https://github.com/shubhamchandak94/nanopore_dna_storage.

Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson’s disease cohort

IntroductionBi-allelic mutations in the gene for glucocerebrosidase (GBA) cause Gaucher disease, an autosomal recessive lysosomal storage disorder. Gaucher disease causing GBA mutations in the heterozygous state are also high risk factors for Parkinson’s disease (PD). GBA analysis is challenging due to a related pseudogene and structural variations (SVs) that can occur at this locus. We have applied and refined a recently developed nanopore DNA sequencing method to analyze GBA variants in a clinically assessed New Zealand longitudinal cohort of PD.MethodWe examined amplicons encompassing the coding region of GBA (8.9kb) from 229 PD cases and 50 healthy controls using the GridION nanopore sequencing platform, and Sanger validation.ResultsWe detected 23 variants in 21 PD cases (9.2% of patients). We detected modest PD risk variant p.N409S (rs76763715) in one case, p.E365K (rs2230288) in 12 cases, and p.T408M (rs75548401) in seven cases, one of whom also had p.E365K. We additionally detected the possible risk variants p.R78C (rs146774384) in one case, p.D179H (rs147138516) in one case which occurred on the same haplotype as p.E365K, and one novel variant c.335C>T or p.(L335=), that potentially impacts splicing of GBA transcripts. Additionally, we found a higher prevalence of dementia among patients with GBA variants.ConclusionThis work confirmed the utility of nanopore sequencing as a high-throughput method to identify known and novel GBA variants, and to assign precise haplotypes. Our observations may contribute to improved understanding of the effects of variants on disease pathogenesis, and to the development of more targeted treatments.