High resolution copy number inference in cancer using short-molecule nanopore sequencing

Abstract

Genome copy number is an important source of genetic variation in health and disease. In cancer, Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms, limiting CNA inference accuracy. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that short-molecule nanopore sequencing reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for short-molecule nanopore sequencing with applications in research and medicine, which includes but is not limited to, CNAs.

Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

“…However, there have been several studies to take advantage of long-read sequencing instruments in sequencing shorter molecules. Some assays [OCEAN, MAS-Iso-Seq] work by either concatenating (8, 28) or otherwise preparing(29) short molecules for sequencing on the PacBio or ONT instrument. While these assays can generate more short reads, they either have to contend with the high cost of the PacBio Sequel IIe sequencer, or the low per-base accuracy of raw ONT reads which even with the latest guppy5 algorithm is only 96% in our hands.…”

Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing

“…ichorCNA analysis. Samtools (Version 1.9) was used to filter BAM alignments, unmapped reads, secondary and supplementary reads, reads with mapping quality less than 20 as in (44), and reads longer than 700bp. For Illumina alignments we trimmed all 'N' nucleotides from the 3' ends of fastq data, alignments were performed to GCF_000001405.39_GRCh38.p13 with BWA mem (43), duplicates were marked using picard MarkDuplicates and removed with samtools;read pairs without the properly-paired flag were removed.Pipelines used for preprocessing and filtering of both Nanopore and Illumina data are available at https://github.com/Puputnik/Fragmentomics_GenomBiol.…”

“…ONT has primarily been used for long-read sequencing, but recent work by our group and others has shown that it can be adapted for short fragments without additional processing steps (16)(17)(18). As an added benefit, the ability to capture much longer cfDNA fragments than shortread sequencing may lead to new discoveries or biomarkers, as was demonstrated recently in the case of longer fragments during pregnancy (19).…”

Comprehensive Structural Variant Detection: From Mosaic to Population-Level

“…Given that this type of variant impacts the greatest number of nucleotides in a genome, it is not surprising that evidence is mounting regarding their importance across all categories of life. This starts e.g., with important speciation events 3 and impacts plants 4,5 , but goes further across human diseases (Mendelian 6,7 as well as complex diseases [8][9][10] ) to cancer development [11][12][13] (e.g., HLA loss, oncogene amplification). Despite the importance of SVs, we are still struggling to detect germline vs. somatic SVs or even robustly identify de novo SVs [14][15][16] .…”