Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED

Abstract

Conventional targeted sequencing methods eliminate many of the benefits of nanopore sequencing, such as the ability to accurately detect structural variants (SVs) or epigenetic modifications. The ReadUntil method allows nanopore devices to selectively eject reads from pores in real-time, which could enable purely computational targeted sequencing. However this requires rapid identification of on-target reads, and most mapping methods require computationally intensive basecalling. We present UNCALLED ( github.com/skovaka/UNCALLED ), an open-source mapper that rapidly matches streaming nanopore current signals to a reference sequence. UNCALLED probabilistically considers k-mers that the signal could represent, and then prunes the candidates based on the reference encoded within an FM-index. We used UNCALLED to deplete sequencing of known bacterial genomes within a metagenomics community, enriching the remaining species by 4.46 fold. UNCALLED also enriched 148 human genes associated with hereditary cancers to 29.6x coverage using one MinION flowcell, enabling accurate detection of SNPs, indels, SVs, and methylation in these genes.

Utilizing nanopore sequencing technology for the rapid and comprehensive characterization of eleven HLA loci; addressing the need for deceased donor expedited HLA typing

“…The Omixon company is in the process of further optimizing its multiplex PCR protocol; we look forward to its most recent improvements. We are also interested in exploring emerging techniques to apply during sequencing to mitigate the effects of locus imbalance with adaptive subsampling during sequencing, which may also further reduce the sequencing time [ 34 ].…”

Nanopanel2 calls phased low-frequency variants in Nanopore panel sequencing data

“…Additionally, we observed good variant calling performance (no FN and only 3 FP over 7 replicates) on a lowly-covered oncospan amplicon (KIT 1) with a median coverage of only 330 reads in the 20k samples (Sup Table S2). Together, this suggests that np2 does not require high read depth to accurately call somatic variants and indicates its possible application in targeted Nanopore sequencing 15, 16 .…”

The Future of Livestock Management: A Review of Real-Time Portable Sequencing Applied to Livestock

“…One possible solution to this limitation is the upcoming release of adaptive sequencing by ONT [ 56 ]. Adaptive or read-until sequencing describes the selective sequencing of particular genomic regions by reversing the flow of ions through specific nanopores [ 56 , 57 ]. Through this, non-target DNA molecules can be ejected from the nanopore increasing the number of available pores for target sequences.…”

“…Through this, non-target DNA molecules can be ejected from the nanopore increasing the number of available pores for target sequences. This allows enrichment of regions to a desired coverage without additional library preparation procedures [ 56 , 57 ]. By using graphics processing unit (GPU)-compatible base calling, sequences can be base called in real time and either rejected or allowed to continue sequencing [ 56 ].…”

“…By using graphics processing unit (GPU)-compatible base calling, sequences can be base called in real time and either rejected or allowed to continue sequencing [ 56 ]. Another approach by Kovaka, et al [ 57 ] involves the targeting of k-mers with an estimated similar electrical signal to the desired enrichment regions. This eliminates the need for GPU base calling which could potentially lend itself towards regional applications of the MinION with adaptive sequencing.…”