Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of long-read sequencing projects, we review the current landscape of available tools and present an online interactive database, long-read-tools.org, to facilitate their browsing. We further focus on the principles of error correction, base modification detection, and long-read transcriptomics analysis and highlight the challenges that remain.

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Opportunities and challenges in long-read sequencing data analysis.

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions 15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads. High-fidelity reads improve variant detection and genome assembly on the PacBio platform.

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Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome.

Structural and quantitative chromosomal rearrangements, collectively referred to as structural variation (SV), contribute to a large extent to the genetic diversity of the human genome and thus are of high relevance for cancer genetics, rare diseases and evolutionary genetics. Recent studies have shown that SVs can not only affect gene dosage but also modulate basic mechanisms of gene regulation. SVs can alter the copy number of regulatory elements or modify the 3D genome by disrupting higher-order chromatin organization such as topologically associating domains. As a result of these position effects, SVs can influence the expression of genes distant from the SV breakpoints, thereby causing disease. The impact of SVs on the 3D genome and on gene expression regulation has to be considered when interpreting the pathogenic potential of these variant types.

/pdf/structural-variation-in-the-3d-genome-20w9u4o4p6.pdf

Structural variation in the 3D genome.

Nanopore sequencing is a rapidly maturing technology delivering long reads in real time on a portable instrument at low cost. Not surprisingly, the community has rapidly taken up this new way of sequencing and has used it successfully for a variety of research applications. A major limitation of nanopore sequencing is its high error rate, which despite recent improvements to the nanopore chemistry and computational tools still ranges between 5% and 15%. Here, we review computational approaches determining the nanopore sequencing error rate. Furthermore, we outline strategies for translation of raw sequencing data into base calls for detection of base modifications and for obtaining consensus sequences.

/pdf/from-squiggle-to-basepair-computational-approaches-for-38jiies7ey.pdf

From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy.

RNA modifications have been historically considered as fine-tuning chemo-structural features of infrastructural RNAs, such as rRNAs, tRNAs, and snoRNAs. This view has changed dramatically in recent years, to a large extent as a result of systematic efforts to map and quantify various RNA modifications in a transcriptome-wide manner, revealing that RNA modifications are reversible, dynamically regulated, far more widespread than originally thought, and involved in major biological processes, including cell differentiation, sex determination, and stress responses. Here we summarize the state of knowledge and provide a catalog of RNA modifications and their links to neurological disorders, cancers, and other diseases. With the advent of direct RNA-sequencing technologies, we expect that this catalog will help prioritize those RNA modifications for transcriptome-wide maps.

The RNA modification landscape in human disease.

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.

/pdf/mapping-and-phasing-of-structural-variation-in-patient-4ohzukh8sn.pdf

Mapping and phasing of structural variation in patient genomes using nanopore sequencing

Structural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications

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Mircea Cretu Stancu

Papers

Mapping and phasing of structural variation in patient genomes using nanopore sequencing

Mapping And Phasing Of Structural Variation In Patient Genomes Using Nanopore Sequencing