lNGS

Long-read Next-Generation Sequencing (lNGS) refers to sequencing technologies that generate reads significantly longer than those produced by traditional short-read platforms. It encompasses methods such as Pacific Biosciences single-molecule real-time sequencing and Oxford Nanopore Technologies nanopore sequencing. Longer reads facilitate de novo genome assembly, resolution of repetitive regions, haplotype phasing, and more accurate detection of structural variants, complementing short-read NGS approaches.

The primary technologies include PacBio SMRT sequencing, which produces reads that can range from several kilobases to tens of kilobases, with recent HiFi or circular consensus reads achieving high base accuracy. Oxford Nanopore sequencing uses nanopores to read DNA or RNA as it passes through a pore, enabling ultra-long reads that can exceed hundreds of kilobases and providing real-time data, including direct detection of certain base modifications.

Applications of lNGS span de novo genome assembly and improving reference genomes, resolving complex structural variations, and phasing and haplotyping large genomic regions. In transcriptomics, full-length isoform sequencing (Iso-Seq) captures complete transcript structures. Epigenetic analyses can leverage direct methylation detection from nanopore data or kinetics-based signals from PacBio. Metagenomics and clinical genomics are also expanding, with lNGS helping to characterize pathogens, microbial communities, and cancer genomes in ways that short reads alone cannot.

Data analysis for lNGS involves specialized workflows, including long-read assemblers (such as Flye, Canu, Shasta), read correction and polishing (Racon, Medaka), and alignment with minimap2. Variant detection and structural variant callers are adapted to long reads, and hybrid approaches combine long and short reads to optimize accuracy and cost. While lNGS can be more expensive per base and demands higher-quality DNA, its ability to span large genomic features makes it a valuable complement to short-read sequencing in research and increasingly in clinical settings.