Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology

Abstract

Screening for genomic sequence variants in genes of predictive and prognostic significance is an integral part of precision medicine. Next-generation sequencing (NGS) technologies are progressively becoming platforms of choice to facilitate this, owing to their massively parallel sequencing capability, which can be used to simultaneously screen multiple markers in multiple samples for a variety of variants (single nucleotide and multi nucleotide variants, insertions and deletions, gene copy number variations, and fusions). A crucial step in the workflow of targeted NGS is the enrichment of the genomic regions of interest to be sequenced, against the whole genomic background. This ensures that the NGS effort is focused to predominantly screen target regions of interest with minimal off-target sequencing, making it more accurate and economical. Polymerase chain reaction-based (PCR, or amplicon-based) and hybridization capture-based methodologies are the two prominent approaches employed for target enrichment. This review summarizes the basic principles of target enrichment utilized by these methods, their multiple variations that have evolved over time, automation approaches, overall comparison of their advantages and drawbacks, and commercially available choices for these methodologies.

Transcriptional profiling during infection of potato NLRs and Phytophthora infestans effectors using cDNA enrichment sequencing

“…Targeted enrichment sequencing ensures minimal off-target sequencing and provides sufficient read depth and sequence coverage for identification of low expressed target genes [46]. In the present study, we observed that 89.98% of RNAseq reads and 76.99% of RenSeq reads map to the potato reference transcriptome.…”

T-Cell Receptor Repertoire Sequencing in the Era of Cancer Immunotherapy

“…An important first step of NGS library preparation is targeted enrichment of transcripts to be sequenced (21). Multiplex polymerase chain reaction (multiplex PCR) and Rapid Amplification of cDNA Ends (5' RACE) are two commonly used methods for targeted amplification during T cell repertoire library preparation.…”

“…Furthermore, primer bias within multiplex PCR risks uneven amplification of alleles, leading to inaccurate relative TCR sequence frequencies (8). Dual barcoding and unique molecular identifier (UMI) modifications to standard PCR protocols facilitate the removal of artifacts introduced during PCR amplification and support accurate downstream sequence analysis (21,22).…”

Sequence-based diagnostics and precision medicine in bacterial and viral infections: from bench to bedside

“…Due to the massive parallel reading capacity of NGS, sequencing the entire biome in the sample is possible; however, targeted NGS (tNGS) is currently more commonly used for clinical purposes [21]. For tNGS, a metataxonomic target, such as the 16 s rRNA gene, is enriched before NGS to maximize the accuracy and efficacy of the assay [22].…”