Single nucleotide variants (SNVs) and phasing
Single nucleotide variants (SNVs) have been widely studied for their associations with phenotypic variation and disease; they are also used to phase haplotypes. However, the use of traditional sequencing technology requires PCR, limiting SNV detection to regions amenable to amplification, and short reads make phasing challenging to resolve. With nanopore sequencing, PCR is not necessary, revealing single-nucleotide polymorphisms (SNPs) in regions inaccessible to other technologies, and phasing is greatly simplified.
- Phase SNVs and resolve compound heterozygosity using long sequencing reads
- Perform real-time analysis with high precision and recall
- Scale to your requirements with a range of nanopore sequencing platforms
Enhance variant calling and phasing with long sequencing reads
Array-based and short-read sequencing analyses of SNVs have shed light on the association between SNVs and the inheritance of complex diseases. However, data from such technologies is only available when a run is completed, which can prevent a rapid response in situations where speed may be vital, such as in outbreak investigations, forensic analysis, and clinical research. Real-time sequencing and analysis with Oxford Nanopore Technologies provide rapid sample-to-genotype, enabling an immediate response. The portable MinION sequencing device is ideal for variant calling in smaller genomes, and for targeted detection, both in the laboratory and in the field. The technology is also scalable: high-throughput variant calling in larger genomes is available in singleplex or samples can be multiplexed with GridION and PromethION.
Assigning a variant to the maternal or paternal chromosome is important for understanding inheritance patterns, mosaicism, and parental origin of de novo mutations, for example. To directly resolve the haplotype of two heterozygous SNPs, they both need to be present within the same sequencing read. This is inherently challenging with short sequencing reads. Nanopore long reads provide sequence context and enhance the phasing of variants (Figure 1).
Nanopore sequencing enables genome-wide variant calling with high precision and recall
Traditional short-read-based methods for SNV calling require PCR, which can introduce bias, and prevents investigation into genomic regions that are not amenable to amplification. With Oxford Nanopore, native DNA strands can be sequenced, negating the requirement for PCR; this enables greater breadth of genome coverage for SNV calling and unambiguous mapping in repetitive regions, as well as access to methylation information from the same sequencing run as standard.
Genomic variants can be characterised with high accuracy using nanopore sequencing data. This is summarised by F1 score, the harmonic mean of precision and recall, in Figure 2, where we display the latest Oxford Nanopore benchmarking metrics for SNP and indel calling in the human genome. The development and optimisation of variant calling tools is a very active area of research. Different bioinformatics tools for SNP calling show variation in performance, and therefore it is important to optimise filtering parameters of the analysis workflow to achieve the best sensitivity and specificity.
Phasing analysis of lung cancer genomes using long nanopore reads
Approximately 20–30% of lung cancer patients remain undiagnosed with respect to their cancerous mutations; variant detection is particularly pressing in cases where an effective treatment remains elusive. To investigate the potential of long nanopore reads for cancer variant analysis, Sakamoto et al. sequenced cancer cell lines and lung cancer research samples and performed integrated analysis of phased genomic, epigenomic, and transcriptomic aberrations. Read the paper to find out how they were able to reveal allele-specific information by phasing data into long phase-blocks with direct methylation calling generated from nanopore sequencing.
Utilising long nanopore reads in carrier screening
Carrier screening (CS) is used to determine if a person is a carrier of a recessive gene and the risk of this being passed onto their children and causing a genetic disorder. Currently, CS requires multiple assays across different platforms to detect SNVs as they are often within long, repetitive regions, making them challenging to resolve using short-read sequencing. Find out how Bradley Hall and his team (Asuragen, USA) are developing an efficient nanopore sequencing workflow to perform CS on a single platform to simplify the workflow for faster results.
How can I call and phase SNVs/SNPs with nanopore sequencing?
To perform genome-wide small variant calling with nanopore technology, we recommend sequencing a whole human genome on the PromethION, or sequencing on the MinION or GridION for targeted analyses. Libraries can be prepared using the Ligation Sequencing Kit, for high throughput and long reads, without amplification.
We recommend wf-human-variation for calling single nucleotide variants and indels. However, other analysis tools are also available from the Community.
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