![]() ![]() The data that these platforms produce differ qualitatively from second-generation sequencing, thus necessitating tailored analysis tools. SMRT and nanopore sequencing technologies were commercially released in 20, respectively, and since then have become suitable for an increasing number of applications. We henceforth refer to these simply as SMRT and nanopore sequencing. Two technologies currently dominate the long-read sequencing space: Pacific Biosciences’ (PacBio) single-molecule real-time (SMRT) sequencing and Oxford Nanopore Technologies’ (ONT) nanopore sequencing. These capabilities, together with continuing progress in accuracy, throughput, and cost reduction, have begun to make long-read sequencing an option for a broad range of applications in genomics for model and non-model organisms. Furthermore, long-read sequencing of native molecules, both DNA and RNA, eliminates amplification bias while preserving base modifications. Long reads can thus improve de novo assembly, mapping certainty, transcript isoform identification, and detection of structural variants. However, natural nucleic acid polymers span eight orders of magnitude in length, and sequencing them in short amplified fragments complicates the task of reconstructing and counting the original molecules. Short-read sequencing is cost-effective, accurate, and supported by a wide range of analysis tools and pipelines. While short-read sequencers such as Illumina’s NovaSeq, HiSeq, NextSeq, and MiSeq instruments BGI’s MGISEQ and BGISEQ models or Thermo Fisher’s Ion Torrent sequencers produce reads of up to 600 bases, long-read sequencing technologies routinely generate reads in excess of 10 kb. Long-read sequencing, or third-generation sequencing, offers a number of advantages over short-read sequencing.
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