eXNVerify: coverage analysis for long and short-read sequencing data in clinical context

Accurate identification of genetic variants to a large extent is based on type of experimental technology, quality of the material and coverage of obtained sequencing data. Our motivation was to create a tool that will evaluate genome coverage and accelerate the introduction of long-read sequencing to medical diagnostics and clinical practice. Here we present eXNVerify: a tool for inspection of clinical data in the context of pathogenic variants. The tool calculates Clinical Depth Coverage – a measure of coverage which we introduce to evaluate loci with pathogenic germline and somatic variants reported in ClinVar. The tool additionally provides visualization options for user-defined genes of interest. Finally, we present an examples of BRCA1, TP53, CFTR application and results of a test conducted in the Extensive Sequence Dataset of Gold-Standard Samples for Benchmarking and Development.

Limitations of lymphoblastoid cell lines for establishing genetic reference datasets in the immunoglobulin loci

Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on short read sequencing data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n = 2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs and short read data for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions.

A high-throughput multiplexing and selection strategy to complete bacterial genomes

Background Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. Results Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

Drastic mutations can be hidden in short-read mapping: thousands of mutation clusters in human genome are explained by short-range template switching

Variation within human genomes is distributed unevenly and variants show spatial clustering. DNA-replication related template switching is a poorly known mutational mechanism capable of causing major chromosomal rearrangements as well as creating short inverted sequence copies that appear as local mutation clusters in sequence comparisons. We reanalyzed haplotype-resolved genome assemblies representing 25 human populations and multinucleotide variants aggregated from 140,000 human sequencing experiments. We found local template switching to explain thousands of complex mutation clusters across the human genome, the loci segregating within and between populations with a small number appearing as de novo mutations. We developed computational tools for genotyping candidate template switch loci using short-read sequencing data and for identification of template switch events using both short-read data and genotype data. These tools will enable building a catalogue of affected loci and studying the cellular mechanisms behind template switching both in healthy organisms and in disease. Strikingly, we noticed that widely-used analysis pipelines for short-read sequencing data - capable of identifying single nucleotide changes - may miss TSM-origin inversions of tens of base pairs, potentially invalidating medical genetic studies searching for causative alleles behind genetic diseases.

Genome-Guided Discovery of Natural Products through Multiplexed Low-Coverage Whole-Genome Sequencing of Soil Actinomycetes on Oxford Nanopore Flongle

Short-read sequencing of GC-rich genomes such as those from actinomycetes results in a fragmented genome assembly and truncated biosynthetic gene clusters (often 10 to >100 kb long), which hinders our ability to understand the biosynthetic potential of a given strain and predict the molecules that can be produced. The current study demonstrates that contiguous DNA assemblies, suitable for analysis of BGCs, can be obtained through low-coverage, multiplexed sequencing on Flongle, which provides a new low-cost workflow ($30 to 40 per strain) for sequencing actinomycete strain libraries.

PerSVade: Personalized Structural Variation detection in your species of interest

Structural variants (SVs) like translocations, deletions, and other rearrangements underlie genetic and phenotypic variation. SVs are often overlooked due to difficult detection from short-read sequencing. Most algorithms yield low recall on humans, but the performance in other organisms is unclear. Similarly, despite remarkable differences across species genomes, most approaches use parameters optimized for humans. To overcome this and enable species-tailored approaches, we developed perSVade (personalized Structural Variation Detection), a pipeline that identifies SVs in a way that is optimized for any input sample. Starting from short reads, perSVade uses simulations on the reference genome to choose the best SV calling parameters. The output includes the optimally-called SVs and the accuracy, useful to assess the confidence in the results. In addition, perSVade can call small variants and copy-number variations. In summary, perSVade automatically identifies several types of genomic variation from short reads using sample-optimized parameters. We validated that perSVade increases the SV calling accuracy on simulated variants for six diverse eukaryotes, and on datasets of validated human variants. Importantly, we found no universal set of optimal parameters, which underscores the need for species-specific parameter optimization. PerSVade will improve our understanding about the role of SVs in non-human organisms.

rRNA Analysis Based on Long-Read High-Throughput Sequencing Reveals a More Accurate Diagnostic for the Bacterial Infection of Ascites

Traditional pathogenic diagnosis presents defects such as a low positivity rate, inability to identify uncultured microorganisms, and time-consuming nature. Clinical metagenomics next-generation sequencing can be used to detect any pathogen, compensating for the shortcomings of traditional pathogenic diagnosis. We report third-generation long-read sequencing results and second-generation short-read sequencing results for ascitic fluid from a patient with liver ascites and compared the two types of sequencing results with the results of traditional clinical microbial culture. The distribution of pathogenic microbial species revealed by the two types of sequencing results was quite different, and the third-generation sequencing results were consistent with the results of traditional microbial culture, which can effectively guide subsequent treatment. Short reads, the lack of amplification, and enrichment to amplify signals from trace pathogens, and host background noise may be the reasons for the high error in the second-generation short-read sequencing results. Therefore, we propose that long-read-based rRNA analysis technology is superior to the short-read shotgun-based metagenomics method in the identification of pathogenic bacteria.

Extensive gene duplication in Arabidopsis revealed by pseudo-heterozygosity

Background: It is becoming apparent that genomes harbor massive amounts of structural variation, and that this variation has largely gone undetected for technical reasons. In addition to being inherently interesting, structural variation can cause artifacts when short-read sequencing data are mapped to a reference genome. In particular, spurious SNPs (that do not show Mendelian segregation) may result from mapping of reads to duplicated regions. Recalling SNP using the raw reads of the 1001 Arabidopsis Genomes Project we identified 3.3 million heterozygous SNPs (44% of total). Given that Arabidopsis thaliana (A. thaliana) is highly selfing, we hypothesized that these SNPs reflected cryptic copy number variation, and investigated them further. Results: While genuine heterozygosity should occur in tracts within individuals, heterozygosity at a particular locus is instead shared across individuals in a manner that strongly suggests it reflects segregating duplications rather than actual heterozygosity. Focusing on pseudo-heterozygosity in annotated genes, we used GWAS to map the position of the duplicates, identifying 2500 putatively duplicated genes. The results were validated using de novo genome assemblies from six lines. Specific examples included an annotated gene and nearby transposon that, in fact, transpose together. Conclusions: Our study confirms that most heterozygous SNPs calls in A. thaliana are artifacts, and suggest that great caution is needed when analysing SNP data from short-read sequencing. The finding that 10% of annotated genes are copy-number variables, and the realization that neither gene- nor transposon-annotation necessarily tells us what is actually mobile in the genome suggest that future analyses based on independently assembled genomes will be very informative.

Transcriptome innovations in primates revealed by single-molecule long-read sequencing

Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Our transcriptomes reveal thousands of novel transcripts, some of them under active translation, expanding and completing the repertoire of primate gene models. Our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.