Application of third-generation sequencing in cancer research

Abstract

In the past several years, nanopore sequencing technology from Oxford Nanopore Technologies (ONT) and single-molecule real-time (SMRT) sequencing technology from Pacific BioSciences (PacBio) have become available to researchers and are currently being tested for cancer research. These methods offer many advantages over most widely used high-throughput short-read sequencing approaches and allow the comprehensive analysis of transcriptomes by identifying full-length splice isoforms and several other posttranscriptional events. In addition, these platforms enable structural variation characterization at a previously unparalleled resolution and direct detection of epigenetic marks in native DNA and RNA. Here, we present a comprehensive summary of important applications of these technologies in cancer research, including the identification of complex structure variants, alternatively spliced isoforms, fusion transcript events, and exogenous RNA. Furthermore, we discuss the impact of the newly developed nanopore direct RNA sequencing (RNA-Seq) approach in advancing epitranscriptome research in cancer. Although the unique challenges still present for these new single-molecule long-read methods, they will unravel many aspects of cancer genome complexity in unprecedented ways and present an encouraging outlook for continued application in an increasing number of different cancer research settings.

Keywords: alternative splicing; application; cancer genome; epigenome; third-generation sequencing.

Figure 1:

Overview of NGS short-read and TGS long-read methods. (A). In NGS by Illumina technology, DNA is fragmented into manageable sizes, and these fragments are ligated to adapters. After library preparation, individual DNA molecules are sequencing for short reads. Following a sequencing run, raw sequence reads were aligned to a reference genome. (B). In PacBio SMRT sequencing, DNA fragment is ligated to hairpin adapters to form a topologically circular molecule, known as SMRTbell. It is loaded onto a SMRT Cell and bound by a DNA polymerase for sequencing. In ONT sequencing, DNA is tagged with sequencing adapters preloaded with a motor protein on one or both ends. The DNA is combined with tethering proteins and loaded onto the flow cell for sequencing. Following a sequencing run, raw sequence reads were aligned to a reference genome. NGS, next generation sequencing; TGS, third generation sequencing.

Figure 2:

Overview of cancer applications by PacBio and ONT. (A) The development platforms of third generation sequencing (TGS); (B) Sequencing a region with two nearly identical repeats (blue) separated by a unique sequence (green) will generate reads corresponding to the upstream region (yellow) in short-read sequencing. Assembly programs for NGS will assemble these reads into a single contig; (C) Haplotype phasing. SNPs (single nucleotide: A or C) between maternal and paternal alleles; (D) Illustration of the use of direct mapping of long-read to a reference genome to resolve complex structural variations, including insertions, deletions, duplications, and complex variation; (E) Detecting of fusion genes; (F) Long-read single cell sequencing; (G) Base modification in a DNA and RNA molecule (represented by a red cycle) give rise to specific signals in TGS data that can be identified using computational methods; (H) Detecting of ncRNAs, including lncRNA, miRNA, and circRNA; (I) A multitude of mRNA transcript isoforms can be generated from a single gene through alternative intron splicing and alternative polyadenylation. Long-read sequencing covers the entire transcript and detects the whole alternative splicing events; (J) TGS-based DNA/RNA analysis for liquid biopsy; (K) Detecting of exogenous RNAs, including viral RNA and bacterial RNA. NGS: next generation sequencing; PacBio: Pacific Bioscience; Oxford Nanopore Technologies (ONT).