Leveraging the power of long reads for targeted sequencing

Abstract

Long-read sequencing technologies have improved the contiguity and, as a result, the quality of genome assemblies by generating reads long enough to span and resolve complex or repetitive regions of the genome. Several groups have shown the power of long reads in detecting thousands of genomic and epigenomic features that were previously missed by short-read sequencing approaches. While these studies demonstrate how long reads can help resolve repetitive and complex regions of the genome, they also highlight the throughput and coverage requirements needed to accurately resolve variant alleles across large populations using these platforms. At the time of this review, whole-genome long-read sequencing is more expensive than short-read sequencing on the highest throughput short-read instruments; thus, achieving sufficient coverage to detect low-frequency variants (such as somatic variation) in heterogenous samples remains challenging. Targeted sequencing, on the other hand, provides the depth necessary to detect these low-frequency variants in heterogeneous populations. Here, we review currently used and recently developed targeted sequencing strategies that leverage existing long-read technologies to increase the resolution with which we can look at nucleic acids in a variety of biological contexts.

Figure 1.

Long-range PCR enrichment. Primers are designed to flank ROI. PCR can be carried out as single reactions for single targets or with multiple targets in a single PCR reaction. Amplified targets can be optionally size-selected via gel if the target size is known. Amplicons are pooled together before library preparation (prep). (Created with BioRender; https://www.biorender.com/)

Figure 2.

Hybridization-based capture. Biotinylated DNA or RNA guides are designed to be complementary to the ROI. The DNA is fragmented to ∼10 kb and amplified if more mass is needed. Next, the probes bind to the denatured DNA. The probe–ROI complex undergoes a bead-based pulldown to separate the target regions from the rest of the genome. The enriched fragments are amplified and size-selected to maintain the target length. The amplicons are then prepared for long-read sequencing. (Created with BioRender; https://www.biorender.com/)

Figure 3.

In-gel Cas9-cleavage and target-specific electrophoresis purification. Cells are embedded in agarose and lysed in gel, maintaining DNA fragment length. Cas9 cleavage is carried out in gel using guides specific to the ROI. PFGE is used to separate the target(s) from background DNA based on size, which is known a priori. The purified target is then prepared for sequencing on either of the long-read platforms using appropriate adapters/kits. (Created with BioRender; https://www.biorender.com/)

Figure 4.

nCATS. DNA is dephosphorylated to prevent sequencing adapter ligation. Cas9 RNPs with guides specific to the ROI are used to cleave the DNA upstream and downstream from the targets. This exposes phosphate groups at the ends of the target strands to which sequencing adapters are then ligated. Targets are, therefore, preferentially sequenced from a sequencing pool consisting of adapter-bound targets and dephosphorylated nontarget DNA. (Created with BioRender; https://www.biorender.com/)

Figure 5.

Single cut and read-out approaches. Dephosphorylated DNA is split into two separate reactions. crRNA guides are designed in a strand-directed manner with separate guide pools prepared for guides cutting upstream versus downstream from each target. Upstream and downstream guide pools are then used to cleave dephosphorylated DNA in separate reactions. After Cas9 cleavage, both reactions are pooled together, and sequencing adapters are ligated. The prepared library is loaded on nanopore flowcells and sequenced. (Created with BioRender; https://www.biorender.com/)

Figure 6.

Approaches with background reduction. (A) NE and CaBagE. DNA is cleaved upstream and downstream from the ROI (dashed lines represent cut sites) using target-specific crRNPs. Immediately after Cas9 cleavage, 5′ and 3′ exonucleases are used to digest background DNA while target ends are protected by the bound Cas9. Heat incubation is used to dissociate Cas9 from the targets and inactivate the exonucleases before sequencing adapter ligation. The prepared library is loaded on nanopore flowcells and sequenced. (B) Affinity-based Cas9-mediated enrichment (ACME). DNA is dephosphorylated to prevent sequencing adapter ligation and Cas9 RNPs with guides specific to the ROI are used to cleave the DNA upstream and downstream from the target(s). After cleavage, Cas9 remains bound to the nontarget side of the cut sites (PAM-distal end). The Cas9 enzyme has a C-terminal 6 Histidine Tag. HisTag Dynabeads are used to pull down Cas9 and the nontarget fragments bound to it from the sequencing pool. Adapters are then ligated to the exposed phosphate groups at the ends of the target strand(s). The prepared library is sequenced on ONT flowcells. (Created with BioRender; https://www.biorender.com/)

Figure 7.

Amplification-free targeted sequencing on the PacBio platform (PacBio No-Amp). DNA is dephosphorylated to prevent sequencing adapter ligation. Cas9 RNPs with guides specific to the ROI are used to cleave the DNA upstream and downstream from the target(s). Sequencing adapters are ligated to the cleaved products. Since no background reduction has been performed yet, nontarget strands protected by Cas9 on both ends will also likely end up with SMRTbell adapters. Exonucleases are introduced to digest the rest of the background DNA. Only those fragments with SMRTbells attached on both sides survive the exonuclease digestion and make up the sequencing pool that is loaded on to PacBio flowcells. (Created with BioRender; https://www.biorender.com/)