• Copy number variations
• Diagnostic approaches
• Hearing loss
• Structural variations

• Humans
• Hearing Loss/genetics
• Hearing Loss/diagnosis
• Genomic Structural Variation/genetics
• Polymorphism, Single Nucleotide
• Genetic Predisposition to Disease

• Hybrid metagenomic assembly
• Microbial community

• Metagenomics/methods
• Microbiota/genetics
• Metagenome/genetics
• High-Throughput Nucleotide Sequencing/methods
• Bioreactors/microbiology
• Sequence Analysis, DNA/methods
• Software

• CRISPR
• CRISPResso2
• Cas
• Cas9
• Indel
• Nanopore
• Sequencing
• gRNA

• CRISPR-Cas Systems/genetics
• INDEL Mutation
• Animals
• RNA, Guide, CRISPR-Cas Systems/genetics
• Nanopore Sequencing/methods
• Sheep/genetics
• Horses/genetics
• Gene Editing/methods
• Myostatin/genetics

• McGarvie Smith Institute
• NSW Government
• Catalina Stud
• University of Sydney

• Basecalling
• Deep neural network
• Genomics sequencing
• Hardware acceleration
• Machine learning

• Deep Learning
• Sequence Analysis, DNA
• Genomics
• Nucleotides
• DNA/genetics
• Nanopores

• Semiconductor Research Corporation
• Google
• Huawei Technologies
• Intel Corporation
• Microsoft
• VMware
• Xilinx
• Swiss Federal Institute of Technology Zurich

• Correction of sequencing errors
• Genome assembly
• Haplotype specific variation
• Metagenome sequencing
• Third-generation sequencing reads

• Sequence Analysis, DNA
• Algorithms
• High-Throughput Nucleotide Sequencing
• Benchmarking

• 956229 HORIZON EUROPE European Research Council
• 872539 HORIZON EUROPE European Research Council
• Universität Bielefeld (3146)

• North American bison
• albinism
• bison reference genome
• genome variant database
• genomics
• tyrosinase gene

• Animals
• Cattle
• Bison/genetics
• Genome
• Chromosomes
• Mutation
• North America

• National Park Service
• Throlson American Bison Foundation Scholarship
• The Houston Safari Club
• College of Veterinary Medicine and Biomedical Sciences
• Texas A&M University
• Boehringer Ingelheim Veterinary Scholars Program
• Texas A&M College of Veterinary Medicine and Biomedical Sciences

• SQANTI
• isoform discovery
• long-read transcriptomics
• transcript simulation
• transcriptome reconstruction

• R21 HG011280 NHGRI NIH HHS

• Oxford nanopore sequencing
• chimeric reads
• metagenomics
• microbial abundance estimation
• sequence simulation

• Metagenome
• Nanopore Sequencing/methods
• Nanopores
• Sequence Analysis, DNA/methods
• Computer Simulation
• Metagenomics/methods
• Software
• Algorithms
• High-Throughput Nucleotide Sequencing/methods

• R01 HG007182 NHGRI NIH HHS
• Genome Canada and Genome BC
• National Institutes of Health
• University of British Columbia
• Natural Sciences and Engineering Research Council of Canada

• Periploca forrestii
• chloroplast genome
• comparative analysis
• genomic structure
• molecular evolution analysis

• annotation error detection
• fungi
• molecular evolution
• primates
• protein analysis tools
• protein domain analysis
• sequence analysis

• GridION
• Illumina
• Oxford Nanopore Technologies (ONT)
• Sepsis punctum
• gene expression
• novel gene
• reproduction
• sexual selection

• Illumina MiSeq
• Nanopore sequencing, next generation sequencing, bioinformatics
• Oxford Nanopore technology GridION
• SARS-CoV-2

• COVID-19
• Genome, Viral
• High-Throughput Nucleotide Sequencing/methods
• Humans
• SARS-CoV-2/genetics
• Whole Genome Sequencing/methods

• 18S-5.8S-26S
• 5S
• 5S rDNA cluster
• C. briggsae
• Caenorhabditis elegans
• Oxford Nanopore technologies

• Animals
• Caenorhabditis elegans/genetics
• DNA, Ribosomal/genetics
• Genomics
• RNA, Ribosomal, 5S/genetics
• Repetitive Sequences, Nucleic Acid/genetics

• P40 OD010440 NIH HHS
• Hong Kong Baptist University

• Clustering
• Edit-distance
• Single-molecule sequencing

Infectious disease monitoring on Oxford Nanopore Technologies (ONT) platforms offers rapid turnaround times and low cost. Tracking low frequency intra-host variants provides important insights with respect to elucidating within-host viral population dynamics and transmission. However, given the higher error rate of ONT, accurate identification of intra-host variants with low allele frequencies remains an open challenge with no viable computational solutions available. In response to this need, we present Variabel, a novel approach and first method designed for rescuing low frequency intra-host variants from ONT data alone. We evaluate Variabel on both synthetic data (SARS-CoV-2) and patient derived datasets (Ebola virus, norovirus, SARS-CoV-2); our results show that Variabel can accurately identify low frequency variants below 0.5 allele frequency, outperforming existing state-of-the-art ONT variant callers for this task. Variabel is open-source and available for download at: www.gitlab.com/treangenlab/variabel.

Tracking low frequency intra-host variants has helped understanding within-host viral population dynamics and transmission. Precise tracking, however, depends partially on the error rate of the sequencing platforms used. Here, Liu et al. present Variabel, a method to rescue low frequency intra-host variants from Oxford Nanopore Technologies (ONT) platforms and validate their approach on Ebola virus, norovirus, and SARS-CoV-2 datasets.

• Eukaryotic organisms
• Gene-related diseases
• Long-read
• RNA sequencing
• Short-read
• Transcriptomes

• High-Throughput Nucleotide Sequencing/methods
• Humans
• Neoplasms/diagnosis
• Neoplasms/genetics
• Rare Diseases/diagnosis
• Rare Diseases/genetics
• Sequence Analysis, RNA/methods
• Transcriptome/genetics
• Exome Sequencing

• dna sequencing
• data processing
• medical genetics

• alternative splicing
• application
• cancer genome
• epigenome
• third-generation sequencing

The major histocompatibility complex (MHC) plays a critical role in the vertebrate immune system. Accurate MHC typing is critical to understanding not only host fitness and disease susceptibility, but also the mechanisms underlying host‐pathogen co‐evolution. However, due to the high degree of gene duplication and diversification of MHC genes, it is often technically challenging to accurately characterise MHC genetic diversity in non‐model species. Here we conducted a systematic review to identify common issues associated with current widely used MHC typing approaches. Then to overcome these challenges, we developed a long‐read based MHC typing method along with a new analysis pipeline. Our approach enables the sequencing of fully phased MHC alleles spanning all key functional domains and the separation of highly similar alleles as well as the removal of technical artefacts such as PCR heteroduplexes and chimeras. Using this approach, we performed population‐scale MHC typing in the Tasmanian devil (Sarcophilus harrisii), revealing previously undiscovered MHC functional diversity in this endangered species. Our new method provides a better solution for addressing research questions that require high MHC typing accuracy. Since the method is not limited by species or the number of genes analysed, it will be applicable for studying not only the MHC but also other complex gene families.

• MHC genetic diversity
• MHC genotyping
• PacBio sequencing
• amplicon analysis
• major histocompatibility complex

Grass carp (Ctenopharyngodon idella) is one of the most widely cultivated fishes in China. High stocking density can reportedly affect fish growth and immunity. Herein we performed PacBio long-read single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing to evaluate the effects of high stocking density on grass carp transcriptome.

SMRT sequencing led to the identification of 33,773 genes (14,946 known and 18,827 new genes). From the structure analysis, 8,009 genes were detected with alternative splicing events, 10,219 genes showed alternative polyadenylation sites and 15,521 long noncoding RNAs. Further, 1,235, 962, and 213 differentially expressed genes (DEGs) were identified in the intestine, muscle, and brain tissues, respectively. We performed functional enrichment analyses of DEGs, and they were identified to be significantly enriched in nutrient metabolism and immune function. The expression levels of several genes encoding apolipoproteins and activities of enzymes involved in carbohydrate enzymolysis were found to be upregulated in the high stocking density group, indicating that lipid metabolism and carbohydrate decomposition were accelerated. Besides, four isoforms of grass carp major histocompatibility complex class II antigen alpha and beta chains in the aforementioned three tissue was showed at least a 4-fold decrease.

The results suggesting that fish farmed at high stocking densities face issues associated with the metabolism and immune system. To conclude, our results emphasize the importance of maintaining reasonable density in grass carp aquaculture.

The online version contains supplementary material available at 10.1186/s12864-021-07924-4.

• Ctenopharyngodon idella
• High stocking density
• Immune function
• Metabolism
• Transcriptome

• African buffalo (Syncerus caffer)
• East Coast fever
• Theileria parva
• cattle
• molecular epidemiology

• BB/S01375X/1 Biotechnology and Biological Sciences Research Council

• Alternative Splicing
• Animals
• Gene Expression Profiling/methods
• Mice
• Oligodendrocyte Precursor Cells/metabolism
• Polyadenylation
• Protein Isoforms/metabolism
• Software

• R01 DK116954 NIDDK NIH HHS
• R01 GM128193 NIGMS NIH HHS
• Spanish Ministry of Education
• Spanish MINECO
• National Institute of General Medical Sciences
• University of Florida

Next-generation sequencing (NGS) offers the opportunity to sequence millions and billions of DNA sequences in a short period, leading to novel applications in personalized medicine, such as cancer diagnostics or antiviral therapy. Nevertheless, sequencing technologies have different error rates, which occur during the sequencing process. If the NGS data is used for diagnostics, these sequences with errors are typically neglected or a worst-case scenario is assumed. In the current study, we focused on the impact of ambiguous bases on therapy recommendations for Human Immunodeficiency Virus 1 (HIV-1) patients. Concretely, we analyzed the treatment recommendation with entry blockers based on prediction models for co-receptor tropism. We compared three different error handling strategies that have been used in the literature, namely (i) neglection, (ii) worst-case assumption, and (iii) deconvolution with a majority vote. We could show that for two or more ambiguous positions per sequence a reliable prediction is generally no longer possible. Moreover, also the position of ambiguity plays a crucial role. Thus, we analyzed the error probability distributions of existing sequencing technologies, e.g., Illumina MiSeq or PacBio, with respect to the aforementioned error handling strategies and it turned out that neglection outperforms the other strategies in the case where no systematic errors are present. In other cases, the deconvolution strategy with the majority vote should be preferred.

• ATAC-sequencing
• Autoimmunity
• ChIP-sequencing
• Inflammation
• Interferon (IFN)
• Long noncoding RNAs (lncRNAs)
• Macrophages
• RNA-sequencing
• Toll-like receptors (TLRs)

• Animals
• Gene Expression Regulation
• Humans
• Immunity, Innate/genetics
• Mice
• RNA, Long Noncoding/chemistry
• RNA, Long Noncoding/genetics
• RNA, Long Noncoding/metabolism

• R21 AR070973 NIAMS NIH HHS
• T32 GM008646 NIGMS NIH HHS
• U01 IP000017 NCIRD CDC HHS

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. Short-read RNAseq is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigate whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After benchmarking our approach, we analyse individual murine B1a cells using a custom multiplexing strategy. We identify thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identify hundreds of genes expressed across B1a cells that display multiple complex isoforms, including several B cell-specific surface receptors. Our results show that we can identify and quantify complex isoforms at the single cell level.

Short-read RNA-seq is limited in its ability to resolve complex transcript isoforms since it cannot sequence full-length cDNA. Here the authors use Oxford Nanopore MinION and their Mandalorion analysis pipeline to measure complex isoforms in B1a cells.

• CsgG
• HAP1
• Nanopore sequencing
• Oxford Nanopore
• R9.4
• complex genomes
• long read sequencing

• CsgG
• HAP1
• Nanopore sequencing
• Oxford Nanopore
• R9.4
• complex genomes
• long read sequencing

Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications with relevance to complex genomes.

• CsgG
• HAP1
• Nanopore sequencing
• Oxford Nanopore
• R9.4
• complex genomes
• long read sequencing