WYMM Tour: Toronto

Oxford Nanopore Technologies’ pore-based sequencing technology enables the direct sequencing of DNA and RNA molecules and as such it allows for the first time, the simultaneous study of genomes with epigenomes and transcriptomes with epitranscriptomes expanding multi-omics approaches. In this talk I will present a sequencing approach which enables the detection of multiple classes of non-coding RNAs excluded by the current standard approach, alongside natively polyadenylated transcripts. I will show how this approach substantially expands the simultaneous representation of multiple classes of non-coding RNAs, without the need for targeted sequencing, presenting a more comprehensive direct RNA-seq approach for the simultaneous study of epitranscriptomes and transcriptomes.

Within clinical microbiology labs, nanopore sequencing has the potential to be utilized in multiple ways. One important manner in which nanopore sequencing (Oxford Nanopore Technologies) can be used is as a tool for diagnosis of microorganisms from clinical specimens. Implementation of nanopore sequencing within a clinical lab requires appropriate validations and evaluations compared to current diagnostic methods and an ongoing process to ensure high-quality testing and reporting occurs. Some of the uses for nanopore sequencing, and the evaluation processes required for implementation in a clinical lab, are described.

We aimed to develop a single streamlined assay to diagnose Glioma, the most common type of malignant brain tumour. It is diagnosed using biomarkers including chromosomal copy number variations (CNVs) and single nucleotide variations (SNVs). CNVs and SNVs are traditionally tested using different large platforms in large genomic facilities creating delays in testing and increasing cost. While nanopore whole genome sequencing can detect CNVs, it does not allow for enough coverage to accurately detect SNVs. Moreover, raw formalin-fixed paraffin-embedded (FFPE) DNA is not compatible with this technology. To fix both challenges we used a PCR based approach that allows enrichment of the SNV targets while creating new nanopore compatible DNA copies. To counteract the bias that PCR creates with uneven amplification, we used a SNP based method to call the CNVs. All the amplicons for CNVs and SNVs were pooled together in one assay and tested on a small cohort of Glioma samples. Expected CNVs such as losses in chr 1p, 19q and 10 were detected with a loss of heterozygosity pattern. The gain in chr 7 was detected with a 2:1 allele frequency pattern. The SNVs were also concordant with reference results. Finally, the SNV and CNV calling analysis were streamlined using custom shell script, further reducing total turnaround time (including sequencing and data analysis) to less than a day for the entire batch of samples. Initial cost analysis shows <50% of the traditional testing costs for the assay. This work is the first to develop a streamlined single test to detect Glioma CNVs and SNVs using FFPE DNA with nanopore sequencing. The shorter testing time, streamlined workflow, and low assay/capital cost could positively influence the care of brain cancer patients.

Abstract: Over 7% of the human genome is comprised of tandem repeats (TRs) – stretches of DNA sequence repeated adjacent to one another. Tandem repeat expansions (TREs) beyond a pathogenic length are associated with over 60 neurological, neurodevelopmental, and neuromuscular disorders. Larger TREs lead to earlier onset, increased severity, and faster progression of disease. GC-rich TREs can become hypermethylated, a mechanism that is associated with disease pathogenesis. Additionally, interruptions in repeat sequences reduce disease severity and result in later age of disease onset. Repeat size is difficult to characterize using existing molecular assays and short-read sequencing. These methods also do not provide methylation information, and only output limited repeat purity information. Nanopore sequencing overcomes these limitations by providing base-pair sequence resolution and methylation information. We have developed a novel tool to estimate tandem repeat size, methylation (adjacent to, and within the repeat) and sequence composition. Here, we demonstrate methods for tandem repeat characterization using targeted Cas9 sequencing, adaptive sampling, and whole-genome sequencing (WGS) approaches. We have also benchmarked our tool against other existing repeat-sizing tools. The estimated TRE size from our tool is strongly correlated with the known size at pathogenic loci in repeat-disease patient samples (R=0.93, p = 2x10-2), and is up to 95.5% accurate at low read depth (5x reads). Characterizing genome-wide TR sizes from Nanopore WGS reveals a strong correlation between our tool and short-read WGS genotypes (R = 0.93, p = 2x10-7). Our work highlights the benefit of Nanopore sequencing for the characterization of TREs. We suggest optimized parameters for analyzing TREs with Nanopore sequencing, and demonstrate the utility of our approach in sizing large, complex TREs. This method can ultimately result in faster characterization of TREs, more accurate prognosis, and faster clinical diagnosis of tandem-repeat associated diseases.

The development of high-throughput single-cell sequencing has enabled the high-resolution mapping of cellular states and hierarchies across diverse sets of tissue. Nevertheless, while certain studies have targeted breast tissue, most existing single-cell studies have not been designed for addressing the question of what cellular constituents and associated molecular alterations are driving the normal-to-malignant transformation. The primary issue with existing work is that they either (a) only target immune subpopulations, (b) do not generate pairs of tumour and matched normal biopsies from the same patient, and/or (c) use sequencing technologies that exhibit technical biases (e.g. 3’ bias) that often limit analyses to only gene expression and not integrative analysis that spans gene expression, mutation, and isoform calling from the same cell and tissue. To address these limitations, we leverage the new, higher accuracy Nanopore chemistry to perform joint genomic and transcriptomic single-cell profiling of matched tumour and normal samples from HER2-negative breast cancer patients. This lightning talk will briefly overview the generally straightforward workflow for performing long-read sequencing at single-cell resolution.

The Centre for Biodiversity Genomics (CBG) at the University of Guelph leads a large international research program that is cataloguing planetary biodiversity by generating short, standardized DNA sequences (DNA barcoding). This talk will describe two main areas in which Nanopore sequencing is being applied to this mission. (1) We have developed simplified protocols to allow a high level of multiplexing on a single flow cell, and user-friendly software tools tailored to analyze the resultant data. Importantly, these protocols are broadly scalable, enabling the recovery of sequences from thousands of specimens in a run (Flongle) to up to 100,000 specimens (MinION) as could be applied in large core facilities. This dramatically reduces the cost of analysis per specimen for routine sequencing. We envision a distributed network of small labs in low-and-middle-income countries, utilizing Nanopore sequencers with our PCR reagents and protocols to survey local biodiversity. (2) Metabarcoding, i.e., sequencing a pool of DNA from a bulk sample as opposed to individual specimens, has the potential to greatly accelerate biodiversity surveys, but due to the high read-depth and sequence fidelity required has until recently only been possible on short-read NGS platforms such as Illumina, thus generating only partial barcode sequences. Current Nanopore chemistry and analytical protocols now yield sufficient read depth and sequencing fidelity, without limitation on read length, to allow metabarcoding of the standard-length COI marker, enabling the data generated by metabarcoding surveys to be directly compared with the full reference library of known taxa.

The Genome Sciences Centre has a long-standing interest in how complete genomic sequencing alongside transcriptomic analysis of human cancers can be done in a clinically meaningful timeline. Therefore providing a precision medicine platform that can be used to influence therapeutic choice. Our personalized oncogenomics program has so far profiled over one thousand tumours from late-stage patients. Nanopore technologies long-read sequencing has the potential to improve both the speed and accuracy of genetic characterization of human cancers. The longer reads providing the ability to more accurately discover and define large scale structural variation. In addition, the alignment of the longer reads provide a more accurate assessment of regions that are highly repetitive or duplicated. Long read sequencing also provides the ability to determine the epigenetic status and therefore the activation of the underlying genes. The longer reads also provide an ability to phase and haplotype variants allowing a more precise characterization of the somatic variants. Likewise, epigenomic patterns can also be haplotyped allowing a more sophisticated view of promoter activity. Correlating methylation patterns associated with imprinted regions has also provided an opportunity to improve hereditary cancer testing through the rapid paternal assignment of pathogenic alleles.