Application Of Long-read Sequencing Research Articles

The Application of Long-Read Sequencing to Cancer.

Cancer is a multifaceted disease arising from numerous genomic aberrations that have been identified as a result of advancements in sequencing technologies. While next-generation sequencing (NGS), which uses short reads, has transformed cancer research and diagnostics, it is limited by read length. Third-generation sequencing (TGS), led by the Pacific Biosciences and Oxford Nanopore Technologies platforms, employs long-read sequences, which have marked a paradigm shift in cancer research. Cancer genomes often harbour complex events, and TGS, with its ability to span large genomic regions, has facilitated their characterisation, providing a better understanding of how complex rearrangements affect cancer initiation and progression. TGS has also characterised the entire transcriptome of various cancers, revealing cancer-associated isoforms that could serve as biomarkers or therapeutic targets. Furthermore, TGS has advanced cancer research by improving genome assemblies, detecting complex variants, and providing a more complete picture of transcriptomes and epigenomes. This review focuses on TGS and its growing role in cancer research. We investigate its advantages and limitations, providing a rigorous scientific analysis of its use in detecting previously hidden aberrations missed by NGS. This promising technology holds immense potential for both research and clinical applications, with far-reaching implications for cancer diagnosis and treatment.

A long-read sequencing and SNP haplotype-based novel preimplantation genetic testing method for female ADPKD patient with de novo PKD1 mutation

The autosomal dominant form of polycystic kidney disease (ADPKD) is the most common hereditary disease that causes late-onset renal cyst development and end-stage renal disease. Preimplantation genetic testing for monogenic disease (PGT-M) has emerged as an effective strategy to prevent pathogenic mutation transmission rely on SNP linkage analysis between pedigree members. Yet, it remains challenging to establish reliable PGT-M methods for ADPKD cases or other monogenic diseases with de novo mutations or without a family history. Here we reported the application of long-read sequencing for direct haplotyping in a female patient with de novo PKD1 c.11,526 G > C mutation and successfully established the high-risk haplotype. Together with targeted short-read sequencing of SNPs for the couple and embryos, the carrier status for embryos was identified. A healthy baby was born without the PKD1 pathogenic mutation. Our PGT-M strategy based on long-read sequencing for direct haplotyping combined with targeted SNP haplotype can be widely applied to other monogenic disease carriers with de novo mutation.

O34: Application of long-read sequencing and telomere-to-telomere genome assembly unveils complex rearrangements and cryptic breakpoints of Robertsonian translocation and ring chromosomes*

O34: Application of long-read sequencing and telomere-to-telomere genome assembly unveils complex rearrangements and cryptic breakpoints of Robertsonian translocation and ring chromosomes*

Third-Generation Cytogenetic Analysis: Diagnostic Application of Long-Read Sequencing

Copy number variants (CNVs) play important roles in the pathogenesis of several genetic syndromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experimental protocols protract diagnostic times from 3 to 15 days. Nanopore sequencing has the ability to reduce time to results for the detection of CNVs with the same resolution of current state-of-the-art diagnostic tests. Nanopore sequencing was compared to molecular karyotyping for the detection of pathogenic CNVs of seven patients with previously diagnosed causative CNVs of different sizes and cellular fractions. Larger chromosomal anomalies included trisomy 21 and mosaic tetrasomy 12p. Among smaller CNVs, two genomic imbalances of 1.3 Mb, a small deletion of 170 kb, and two mosaic deletions (1.2 Mb and 408 kb) were tested. DNA was sequenced and data generated during runs were analyzed in online mode. All pathogenic CNVs were identified with detection time inversely proportional to size and cellular fraction. Aneuploidies were called after only 30 minutes of sequencing, whereas 30 hours were needed to call small CNVs. These results demonstrate the clinical utility of our approach that allows the molecular diagnosis of genomic disorders within a 30-minute to 30-hour time frame and its easy implementation as a routinary diagnostic tool.

ECNano: A cost-effective workflow for target enrichment sequencing and accurate variant calling on 4800 clinically significant genes using a single MinION flowcell

BackgroundThe application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample.MethodWe introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4800 clinically important genes and regions using a single MinION flowcell. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples.ResultsECNano achieved deep on-target depth of coverage (DoC) at average > 100× and > 98% uniformity using one MinION flowcell. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30× DoC. ECNano obtained an average read length of 1000 bp. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having ≥ 30× DoC. Clair-ensemble achieved > 99% recall and accuracy for SNV calling. The whole workflow from wet-lab protocol to variant detection was completed within three days.ConclusionWe presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4800 clinically significant genes and regions using a single MinION flowcell. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction.

Application of long-read sequencing to elucidate complex pharmacogenomic regions: a proof of principle

The use of pharmacogenomics in clinical practice is becoming standard of care. However, due to the complex genetic makeup of pharmacogenes, not all genetic variation is currently accounted for. Here, we show the utility of long-read sequencing to resolve complex pharmacogenes by analyzing a well-characterised sample. This data consists of long reads that were processed to resolve phased haploblocks. 73% of pharmacogenes were fully covered in one phased haploblock, including 9/15 genes that are 100% complex. Variant calling accuracy in the pharmacogenes was high, with 99.8% recall and 100% precision for SNVs and 98.7% precision and 98.0% recall for Indels. For the majority of gene-drug interactions in the DPWG and CPIC guidelines, the associated genes could be fully resolved (62% and 63% respectively). Together, these findings suggest that long-read sequencing data offers promising opportunities in elucidating complex pharmacogenes and haplotype phasing while maintaining accurate variant calling.

OP336 - Clinical application of long-read sequencing in unsolved rare disease

OP336 - Clinical application of long-read sequencing in unsolved rare disease

Long-read-tools.org: an interactive catalogue of analysis methods for long-read sequencing data.

BackgroundThe data produced by long-read third-generation sequencers have unique characteristics compared to short-read sequencing data, often requiring tailored analysis tools for tasks ranging from quality control to downstream processing. The rapid growth in software that addresses these challenges for different genomics applications is difficult to keep track of, which makes it hard for users to choose the most appropriate tool for their analysis goal and for developers to identify areas of need and existing solutions to benchmark against.FindingsWe describe the implementation of long-read-tools.org, an open-source database that organizes the rapidly expanding collection of long-read data analysis tools and allows its exploration through interactive browsing and filtering. The current database release contains 478 tools across 32 categories. Most tools are developed in Python, and the most frequent analysis tasks include base calling, de novo assembly, error correction, quality checking/filtering, and isoform detection, while long-read single-cell data analysis and transcriptomics are areas with the fewest tools available.ConclusionContinued growth in the application of long-read sequencing in genomics research positions the long-read-tools.org database as an essential resource that allows researchers to keep abreast of both established and emerging software to help guide the selection of the most relevant tool for their analysis needs.

Application of long-read sequencing to the detection of structural variants in human cancer genomes.

In recent years, the so-called long-read sequencing technology has had a substantial impact on various aspects of genome sciences. Here, we introduce recent studies of cancerous structural variants (SVs) using long-read sequencing technologies, namely Pacific Biosciences (PacBio) sequencers, Oxford Nanopore Technologies (ONT) sequencers, and linked-read methods. By taking advantage of long-read lengths, these technologies have enabled the precise detection of SVs, including long insertions by transposable elements, such as LINE-1. In addition to SV detection, the epigenome status (including DNA methylation and haplotype information) surrounding SV loci has also been unveiled by long-read sequencing technologies, to identify the effects of SVs. Among the various research fields in which long-read sequencing has been applied, cancer genomics has shown the most remarkable advances. In fact, many studies are beginning to shed light on the detection of SVs and the elucidation of their complex structures in various types of cancer. In the particular case of cancers, we summarize the technical limitations of the application of this technology to the analysis of clinical samples. We will introduce recent achievements from this viewpoint. However, a similar approach will be started for other applications in the near future. Therefore, by complementing the current short-read sequencing analysis, long-read sequencing should reveal the complex nature of human genomes in their healthy and disease states, which will open a new opportunity for a better understanding of disease development and for a novel strategy for drug development.

Implications of Error-Prone Long-Read Whole-Genome Shotgun Sequencing on Characterizing Reference Microbiomes.

SummaryLong-read sequencing techniques, such as the Oxford Nanopore Technology, can generate reads that are tens of kilobases in length and are therefore particularly relevant for microbiome studies. However, owing to the higher per-base error rates than typical short-read sequencing, the application of long-read sequencing on microbiomes remains largely unexplored. Here we deeply sequenced two human microbiota mock community samples (HM-276D and HM-277D) from the Human Microbiome Project. We showed that assembly programs consistently achieved high accuracy (∼99%) and completeness (∼99%) for bacterial strains with adequate coverage. We also found that long-read sequencing provides accurate estimates of species-level abundance (R = 0.94 for 20 bacteria with abundance ranging from 0.005% to 64%). Our results not only demonstrate the feasibility of characterizing complete microbial genomes and populations from error-prone Nanopore sequencing data but also highlight necessary bioinformatics improvements for future metagenomics tool development.

Annotated bacterial chromosomes from frame-shift-corrected long-read metagenomic data

BackgroundShort-read sequencing technologies have long been the work-horse of microbiome analysis. Continuing technological advances are making the application of long-read sequencing to metagenomic samples increasingly feasible.ResultsWe demonstrate that whole bacterial chromosomes can be obtained from an enriched community, by application of MinION sequencing to a sample from an EBPR bioreactor, producing 6 Gb of sequence that assembles into multiple closed bacterial chromosomes. We provide a simple pipeline for processing such data, which includes a new approach to correcting erroneous frame-shifts.ConclusionsAdvances in long-read sequencing technology and corresponding algorithms will allow the routine extraction of whole chromosomes from environmental samples, providing a more detailed picture of individual members of a microbiome.

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections.

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 genes. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving variant differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads–the earliest in infection that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse gene expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and the ability to identify mosaic gene formation early during the infection process.

A larval form of Triarthrus.

<h3>Abstract</h3> <h3>Background</h3> The application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample. <h3>Method</h3> We introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4,800 clinically important genes and regions using a single MinION flowcell. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples. <h3>Results</h3> ECNano achieved deep on-target depth of coverage (DoC) at average &gt;100x and &gt;98% uniformity using one MinION flowcell. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30x DoC. ECNano obtained an average read length of 1,000 bp. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having ≥ 30x DoC. Clair-ensemble achieved &gt;99% recall and accuracy for SNV calling. The whole workflow from wet-lab protocol to variant detection was completed within three days. <h3>Conclusion</h3> We presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4,800 clinically significant genes and regions using a single MinION flowcell. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction.