Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers

Abstract

Targeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost. We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined ‘target-capture-PCR’ workflow. This approach uses physically linked capture probes and PCR primers and is expected to work with panel sizes from 100 bp to >10 Mbp. It reduces the time and complexity of the capture workflow, eliminates long hybridization and wash steps and enables rapid library construction and target capture. High on-target read fractions are achievable due to repeated sequence selection in the target-capture-PCR step, thus lowering sequencing cost. We have demonstrated this technology on sample types including cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) derived DNA, capturing a 35-gene pan-cancer panel, and therein detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), variants as low as 0.25% abundance were detected, limited by input mass and sequencing depth. Additionally, sequencing libraries were prepared in less than eight hours from extracted DNA to loaded sequencer, demonstrating that LTC holds promise as a broadly applicable tool for rapid, cost-effective and high performance targeted sequencing.

Current challenges and best-practice protocols for microbiome analysis

“…Most marker genes are functionally conserved across phylogenetic distances and thus also serve as a molecular clock for studying evolutionary transitions and changes. The most commonly used target gene for bacterial identification is 16S rRNA (or 16S rDNA), which is the gold standard in microbial typing [ 15 , 16 ]. The 16S rRNA gene encodes prokaryotic small 30S subunit of the 70S ribosomal complex in most bacteria and archaea.…”

Next Generation Sequencing for Gene Fusion Analysis in Lung Cancer: A Literature Review

“…DNA-or RNA-targeted panels for gene fusions can be hybrid-capture-or amplicon-based (Table 1). The hybrid-capture method implies a gene-specific enrichment by a hybridization step with probes complementary to the regions of interest, whereas amplicon-based methods rely on a multiplex PCR (mPCR) with primers specific for each target (Figure 1) [20][21][22].…”

“…Classical mPCR is based on the use of primers flanking exon-exon fusion combinations allowing to detect only known fusion variants (Figure 1). However, RNA-based fusion panels also include testing for expression imbalances between 5 and 3 regions of the target genes, thus permitting an identification of the presence of a rearrangement even if the fusion partner is not known and is not included in the panel [21,29]. The amplicon-based approach is one of the most common approaches for gene fusion analysis and different commercial and custom panels have already been tested and validated.…”

“…The hybrid-capture method implies a gene-specific enrichment by a hybridization step with biotinylated DNA or RNA probes specific for the regions of interest (Figure 1). If DNA is analyzed, probes are complementary to intronic, exonic and intergenic regions, whereas if RNA is analyzed, probes target only exonic regions [9,21]. This approach allows to identify known and novel fusion variants for any gene targeted by the capture panel; obviously completely novel fusion genes are omitted, indeed at least one of the fusion partners has to be present on the target panel [22].…”

“…This approach uses physically linked capture probes and PCR primers, it has been demonstrated that this technology can work on different sample types including ctDNA and FFPE-derived DNA, capturing a 35-gene pan-cancer panel, and detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), the y demonstrated that variants with as low as 0.25% abundance could be detected [21].…”