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.
Highlights
Copy number variation is a common form of genomic variation in humans and has been found to be correlated with a number of pathologies including rare genomic disorders [1], neurological diseases [2,3] and cancer [4]
The number of reads/molecules returned in a standard long read MinION run is limited by (i) the design of the nanopore array, especially the overall number of channels and the overall number of active pores; (ii) the molecule kinetics of DNA fragments docking to the nanopore, measured by the duration between the end of one read and the beginning of the within the same pore and (iii) the residence time of a molecule translocating through a given pore
We hypothesized that loading DNA molecules that are shorter in length (e.g. 400 bp in median length compared to a standard 10 kb) would decrease the residence time of each DNA molecule in a pore and facilitate, over a period of time, the translocation of more DNA molecules and the retrieval of higher read/molecule counts
Summary
Copy number variation is a common form of genomic variation in humans and has been found to be correlated with a number of pathologies including rare genomic disorders [1], neurological diseases [2,3] and cancer [4]. Generation sequencing addresses these limitations by showing that high resolution CNA information is retrievable from short-read sequencing data using a variety of experimental and computational approaches [8,9,10,11,12,13]. These efforts have culminated in the successful implementation of shortread sequencing in the clinic [14,15]. Community-based centers with the ability to retrieve cancer sequence information, in a cost-effective manner, is likely to enhance the quality of health care overall
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