Beyond assembly: the increasing flexibility of single-molecule sequencing technology

Abstract

The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read sequencing has emerged as an essential tool in deciphering genome structure and function, including filling gaps in the human reference genome, measuring the epigenome and characterizing splicing variants in the transcriptome. With recent technological developments, these single-molecule technologies have moved beyond genome assembly and are being used in a variety of ways, including to selectively sequence specific loci with long reads, measure chromatin state and protein-DNA binding in order to investigate the dynamics of gene regulation, and rapidly determine copy number variation. These increasingly flexible uses of single-molecule technologies highlight a young and fast-moving part of the field that is leading to a more accessible era of nucleic acid sequencing.

Fig. 1. Long-read targeted sequencing methods.

Long-read targeted enrichment methods fall within broad categories including PCR enrichment, hybridization capture, Cas-mediated enrichment and adaptive sampling. a, PCR enrichment uses specific primers to amplify regions of interest before library preparation. b, Hybridization capture uses biotinylated antisense probes designed against regions of interest to isolate DNA fragments containing the targets. PCR and hybridization capture enrichment methods are both commonly used with short-read sequencing and have been adapted to long-read sequencing. c, Cas-mediated enrichment uses Cas ribonuclear complexes (most commonly Cas9) to cut on either side of regions of interest. Cut fragments are selectively sequenced owing to preferential adapter ligation to the freshly cut ends. Targeted fragments can be further enriched through depletion of off-target fragments^–. d, Enrichment using adaptive sampling is a nanopore sequencing method in which regions of interest are selectively sequenced by controlling the voltage at individual pores to eject unwanted fragments. ONT, Oxford Nanopore Technologies.

Fig. 2. Long-read, single-molecule methyltransferase footprinting methods can reveal heterogeneity and coordination of chromatin states.

a, In methyltransferase footprinting assays, a methyltransferase enzyme deposits exogenous methylation on accessible DNA, which may include linker DNA between histones, open chromatin regions or regions surrounding transcription factors bound to DNA. b, When this exogenous labelling is performed on long, single molecules, the heterogeneity of nucleosome positioning, open or closed chromatin and protein–DNA binding can be measured on single molecules. c, With long molecules that span multiple regulatory elements, the coordination between adjacent sites can be measured, potentially revealing unknown aspects of gene regulation. d, Antibody-directed methyltransferase labelling builds on methyltransferase footprinting by concentrating labelling around binding sites of specific proteins. The methyltransferase is fused to protein A, protein G or both, which bind to IgG antibodies.

Fig. 3. Applications of shorter-read sequencing (<5 kb) on single-molecule platforms.

a, Short reads can be quickly sequenced on portable Oxford Nanopore sequencing devices, returning real-time information about copy number variants and aneuploidy in 3 h or less. b, Primary sequence, fragment patterns and endogenous methylation can be measured simultaneously with single-molecule platforms, and that information can be used to assign reads to tissues of origin. c, Accuracy of short reads on single-molecule platforms can be improved by correcting for errors by reading the same molecule multiple times. d, The cost of sequencing short fragments on single-molecule platforms can be decreased by combining multiple different short molecules into a single, long molecule.