Abstract 2279: Cas9 based targeted nanopore sequencing helps identify structural variants in breast cancer genes
Authors: Iyer Shruti V. , Kramer Melissa , Goodwin Sara , McCombie W. Richard 
Published: June 2022

Structural variations (SV), a hallmark of genomic instability in cancer, tend to be recurrent & have been associated with several cancer types wherein they either activate oncogenes or inactivate tumor suppressor genes. NGS is mostly blind to large SVs, lacking sensitivity with FPR up to 89% in SV detection. Long-read sequencing (LRS) can address larger variations by generating read lengths of tens of thousands of bases & have helped identify thousands of genomic features pertinent to cancer previously missed by NGS. However, throughput & coverage of whole genome LRS makes it infeasible to conduct large-scale genomic studies to detect rare alleles.

Cancer cells within the same sample can be heterogenous, with subpopulations exhibiting different genomic features, that are difficult to detect with just 30x whole genome sequencing. Targeted sequencing significantly improves accuracy & coverage by offering depths necessary to detect these rare alleles in a heterogenous population of cells. Recently, we developed ACME, an Affinity-based Cas9-Mediated Enrichment approach that is an improvement on nanopore Cas9-targeted sequencing (nCATS) from Oxford Nanopore Technologies with the inclusion of a background reduction step. We targeted 10 prominent cancer genes in MCF 10A & SK-BR-3 breast cell lines with ACME & observed an increase in enrichment & coverage of all genes on the panel, with enrichment as high as 5000-fold for some genes. We achieved a ~ 75-fold enrichment & 35-65x coverage of the BRCA2 region, an important breast cancer gene that was a ~90 kb target on our panel. Across our panel, we found that ACME helps increase the number of single contiguous reads that span the entire target, which ultimately helps with better alignment & SV detection. ACME detected all SVs within our target regions that had been previously inferred by PacBio & ONT whole-genome LRS, but with higher depth. This allows for rare variant detection, making it an effective long-read targeting platform.

We are currently developing ACME + native barcoding, which gave us mean target coverage of 10-30x in initial testing, to enable sample multiplexing along with target multiplexing. Our efforts are also directed towards expanding our cancer gene panel to target 35 genes common between breast, pancreatic, & colorectal cancer. We have also successfully expanded ACME’s use to the PromethION high throughput device (currently unsupported by ONT for nCATS) & have observed a 4-fold increase in coverage when compared to GridION using the same sample and mass for library prep. Performing high throughput targeted LRS on &gt;15 samples per PromethION flowcell would allow SV analysis of several important cancer genes across 100s of samples, helping define the landscape of such variants in the population & identify regions of therapeutic or diagnostic interest.

Citation Format: Shruti V. Iyer, Melissa Kramer, Sara Goodwin, W. Richard McCombie. Cas9 based targeted nanopore sequencing helps identify structural variants in breast cancer genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2279.