Abstract
As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.
Highlights
Next-generation sequencing (NGS) has allowed for the rapid characterization of genomes, exomes and transcriptomes
We determined that physical coverage is directly affected by insert size such that physical coverage increases with longer insert sizes when sequencing a fixed read length
Physical coverage is considered in this analysis because it reflects the size of the insert being sequenced and is associated with our ability to identify copy number variants (CNVs) and translocations
Summary
Next-generation sequencing (NGS) has allowed for the rapid characterization of genomes, exomes and transcriptomes. The cost of performing whole genome sequencing (WGS) has decreased in recent years, it is more costly compared with exome and RNA sequencing (RNAseq) when sequencing to 30Â coverage Owing to this caveat and the existing utility of using deep exome sequencing to identify potentially targetable small somatic events in cancer genomes, the need for identifying an alternative WGS strategy for identifying breakpoints, which characterize structural variants and copy number changes, is clear. Illumina recently released a new Nextera Mate Pair Sample Preparation Kit that requires 1–4 mg of genomic DNA. This approach retains transposomemediated fragmentation that results in an enzymatic footprint that requires trimming of sequencing data, and still requires circularization and biotin pull-down, and decreases the ease of library preparation. An alternative user-friendly strategy that requires lower inputs, that does not require post-sequencing trimming and that allows for increased physical coverage and analysis of regions greater than that accomplished by short insert (SI) sequencing is needed
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