Adapting long read sequencing technologies for targeted and single cell applications

Iyer, S., Goodwin, S., McCombie, W. R. (July 2019) Adapting long read sequencing technologies for targeted and single cell applications. Cancer Research, 79 (13S). p. 5136. ISSN 0008-5472

DOI: 10.1158/1538-7445.am2019-5136


Structural variations (SV), a hallmark of genomic instability in cancer, include insertions, deletions, duplications, inversions, or translocations that can either activate oncogenes or inactivate tumor suppressor genes. These variants contribute to the complexity of the cancer genome but have not been successfully resolved yet. While short-read sequencing has aided cancer genomics, it has performed poorly in SV detection, with false positive and false negative rates of 50% or more. Long-read sequencing generates reads that are >10 kb long and has helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. Long reads can span SV with a single continuous read, giving a clearer idea of the variation, its position, and size. Currently, long-read sequencing methods are greatly limited by the DNA preparation step, with fragment lengths and yield often compromised by standard extraction methods. The potential to generate reads at the megabase level relies greatly on library preps using large amounts (several μg) of DNA input, which prevents its application to samples like patient tumors that are often limited in DNA. Our overarching goal is to develop a targeted approach that generates large contiguous reads from moderate to low input DNA masses. To this end, we use different extraction, amplification, fragmentation, and enrichment strategies to efficiently target and resolve complex regions of the genome that are often associated with disease conditions. Development of a targeted long-read sequencing strategy using low DNA input would give us the potential to explore the landscape of genetic variation across several individuals and heterogeneous cell populations. We are currently employing and adapting CRISPR-based targeting techniques to enrich for specific regions of the genome from intact cells and/or high-molecular-weight DNA. Citation Format: Shruti Iyer, Sara Goodwin, W. Richard McCombie. Adapting long read sequencing technologies for targeted and single cell applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5136.

Item Type: Paper
Additional Information: Meeting Abstract
Subjects: diseases & disorders > cancer
Investigative techniques and equipment > assays > long-read sequencing
CSHL Authors:
Communities: CSHL labs > McCombie lab
Depositing User: Matthew Dunn
Date: July 2019
Date Deposited: 08 Nov 2019 19:19
Last Modified: 08 Nov 2019 19:19
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