Short-read Sequencing Data Research Articles

Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.

Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

One to host them all: genomics of the diverse bacterial endosymbionts of the spider Oedothorax gibbosus.

Bacterial endosymbionts of the groups Wolbachia, Cardinium and Rickettsiaceae are well known for their diverse effects on their arthropod hosts, ranging from mutualistic relationships to reproductive phenotypes. Here, we analysed a unique system in which the dwarf spider Oedothorax gibbosus is co-infected with up to five different endosymbionts affiliated with Wolbachia, 'Candidatus Tisiphia' (formerly Torix group Rickettsia), Cardinium and Rhabdochlamydia. Using short-read genome sequencing data, we show that the endosymbionts are heterogeneously distributed among O. gibbosus populations and are frequently found co-infecting spider individuals. To study this intricate host-endosymbiont system on a genome-resolved level, we used long-read sequencing to reconstruct closed genomes of the Wolbachia, 'Ca. Tisiphia' and Cardinium endosymbionts. We provide insights into the ecology and evolution of the endosymbionts and shed light on the interactions with their spider host. We detected high quantities of transposable elements in all endosymbiont genomes and provide evidence that ancestors of the Cardinium, 'Ca. Tisiphia' and Wolbachia endosymbionts have co-infected the same hosts in the past. Our findings contribute to broadening our knowledge about endosymbionts infecting one of the largest animal phyla on Earth and show the usefulness of transposable elements as an evolutionary 'contact-tracing' tool.

Diverse Microbial Hot Spring Mat Communities at Black Canyon of the Colorado River.

The thermophilic microbial mat communities at hot springs in the Black Canyon of the Colorado River, thought to harbor the protistan human pathogen Naegleria fowleri, were surveyed using both culture-independent and -dependent methods to further understand the ecology of these hot spring microbiomes. Originating from Lake Mead source water, seven spring sites were sampled, varying in temperature from 25 to 55 °C. Amplicon-based high-throughput sequencing of twelve samples using 16S rRNA primers (hypervariable V4 region) revealed that most mats are dominated by cyanobacterial taxa, some but not all similar to those dominating the mats at other studied hot spring systems. 18S rRNA amplicon sequencing (V9 region) demonstrated a diverse community of protists and other eukaryotes including a highly abundant amoebal sequence related to Echinamoeba thermarum. Additional taxonomic and diversity metric analyses using near full-length 16S and 18S rRNA gene sequencing allowed a higher sequence-based resolution of the community. The mat sequence data suggest a major diversification of the cyanobacterial orders Leptolyngbyales, as well as microdiversity among several cyanobacterial taxa. Cyanobacterial isolates included some representatives of ecologically abundant taxa. A Spearman correlation analysis of short-read amplicon sequencing data supported the co-occurrences of populations of cyanobacteria, chloroflexi, and bacteroidetes providing evidence of common microbial co-occurrences across the Black Canyon hot springs.

An assessment of bioinformatics tools for the detection of human endogenous retroviral insertions in short-read genome sequencing data.

There is a growing interest in the study of human endogenous retroviruses (HERVs) given the substantial body of evidence that implicates them in many human diseases. Although their genomic characterization presents numerous technical challenges, next-generation sequencing (NGS) has shown potential to detect HERV insertions and their polymorphisms in humans. Currently, a number of computational tools to detect them in short-read NGS data exist. In order to design optimal analysis pipelines, an independent evaluation of the available tools is required. We evaluated the performance of a set of such tools using a variety of experimental designs and datasets. These included 50 human short-read whole-genome sequencing samples, matching long and short-read sequencing data, and simulated short-read NGS data. Our results highlight a great performance variability of the tools across the datasets and suggest that different tools might be suitable for different study designs. However, specialized tools designed to detect exclusively human endogenous retroviruses consistently outperformed generalist tools that detect a wider range of transposable elements. We suggest that, if sufficient computing resources are available, using multiple HERV detection tools to obtain a consensus set of insertion loci may be ideal. Furthermore, given that the false positive discovery rate of the tools varied between 8% and 55% across tools and datasets, we recommend the wet lab validation of predicted insertions if DNA samples are available.

A highly multiplexed assay to monitor pathogenicity, fungicide resistance and gene flow in the fungal wheat pathogen Zymoseptoria tritici.

Crop pathogens pose severe risks to global food production due to the rapid rise of resistance to pesticides and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in the genetic composition of pathogen populations. Here we report the design of a microfluidics-based amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici. The fungus causes one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. We optimized the primer design by integrating polymorphism data from 632 genomes of the same species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of the short-read sequence data generated by the assay showed a fairly stable success rate across samples to amplify a large number of loci. The performance was consistent between samples originating from pure genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed genotypes, we found that the assay recovers variations in allele frequencies. We also explored the potential of the amplicon assay to recover transposable element insertion polymorphism relevant for fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more sustainable crop protection and yields.

Inference of Polyploid Origin and Inheritance Mode from Population Genomic Data.

Whole-genome duplications yield varied chromosomal pairing patterns, ranging from strictly bivalent to multivalent, resulting in disomic and polysomic inheritance modes. In the bivalent case, homeologous chromosomes form pairs, where in a multivalent pattern all copies are homologous and are therefore free to pair and recombine. As sufficient sequencing data is more readily available than high-quality cytological assessments of meiotic behavior or population genetic assessment of allelic segregation, especially for non-model organisms, bioinformatics approaches to infer origins and inheritance modes of polyploids using short-read sequencing data are attractive. Here we describe two such approaches, where the first is based on distributions of allelic read depth at heterozygous sites within an individual, as the expectations of such distributions are different for disomic and polysomic inheritance modes. The second approach is more laborious and based on a phylogenetic assessment of partially phased haplotypes of a polyploid in comparison to the closest diploid relatives. We discuss the sources of deviations from expected inheritance patterns, advantages and pitfalls of both methods, effects of mating types on the performance of the methods, and possible future developments.

Assembly and Annotation of Viral Metagenomes from Short-Read Sequencing Data.

Viral metagenomics enables the detection, characterization, and quantification of viral sequences present in shotgun-sequenced datasets of purified virus-like particles and whole metagenomes. Next generation sequencing (Illumina) derived short single or paired-end read runs are a principal platform for metagenomics, and assembly of short reads allows for the identification of distinguishing viral signatures and complex genomic features for taxonomy and functional annotation. Here we describe the identification and characterization of viral genome sequences, bacteriophages, and eukaryotic viruses, from a cohort of human stool samples, using multiple methods. Following the purification of virus-like particles, sequencing, quality refinement, and genome assembly, we begin the protocol with raw short reads deposited in an open-source nucleotide archive. We highlight the use of VIBRANT, an automated computational tool for the characterization of microbial viruses and their viral community function. Finally, we also describe an alternative assembly-free option of mapping reads to established databases of reference genomes and previously characterized metagenome-assembled viral genomes.

Reference Genome Sequences of the Oriental Armyworm, Mythimna separata (Lepidoptera: Noctuidae)

Lepidopteran insects are an important group of animals, including those used as biochemical and physiological model species in the insect and silk industries as well as others that are major agricultural pests. Therefore, the genome sequences of several lepidopteran insects have been reported. The oriental armyworm, Mythimna separata, is an agricultural pest commonly used to study insect immune reactions and interactions with parasitoid wasps as hosts. To improve our understanding of these research topics, reference genome sequences were constructed in the present study. Using long-read and short-read sequence data, de novo assembly and polishing were performed and haplotigs were purged. Subsequently, gene predictions and functional annotations were performed. To search for orthologs of the Toll and Immune Deficiency (IMD) pathways and for C-type lectins, annotation data analysis, BLASTp, and Hummer scans were performed. The M. separata genome is 682 Mbp; its contig N50 was 2.7 Mbp, with 21,970 genes and 24,452 coding sites predicted. All orthologs of the core components of the Toll and IMD pathways and 105 C-type lectins were identified. These results suggest that the genome data were of sufficient quality for use as reference genome data and could contribute to promoting M. separata and lepidopteran research at the molecular and genome levels.

Optical genome mapping and revisiting short-read genome sequencing data reveal previously overlooked structural variants disrupting retinal disease−associated genes

PurposeStructural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs. MethodsOptical genome mapping was performed to improve SV detection in short-read genome sequencing−negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria. ResultsIn a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses. ConclusionCritical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.

STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci

Expansions of short tandem repeats (STRs) cause many rare diseases. Expansion detection is challenging with short-read DNA sequencing data since supporting reads are often mapped incorrectly. Detection is particularly difficult for “novel” STRs, which include new motifs at known loci or STRs absent from the reference genome. We developed STRling to efficiently count k-mers to recover informative reads and call expansions at known and novel STR loci. STRling is sensitive to known STR disease loci, has a low false discovery rate, and resolves novel STR expansions to base-pair position accuracy. It is fast, scalable, open-source, and available at: github.com/quinlan-lab/STRling.

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand

BackgroundThe mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated.MethodsThis study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis.ResultsWe successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species.ConclusionsThis was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite.Graphical abstract

Improved Assembly of Metagenome-Assembled Genomes and Viruses in Tibetan Saline Lake Sediment by HiFi Metagenomic Sequencing.

With the development and reduced costs of high-throughput sequencing technology, environmental dark matter, such as novel metagenome-assembled genomes (MAGs) and viruses, is now being discovered easily. However, due to read length limitations, MAGs and viromes often suffer from genome discontinuity and deficiencies in key functional elements. Here, by applying long-read sequencing technology to sediment samples from a Tibetan saline lake, we comprehensively analyzed the performance of high-fidelity (HiFi) reads and the possibility of integration with short-read next-generation sequencing (NGS) data. In total, 207 full-length nonredundant 16S rRNA gene sequences and 19 full-length nonredundant 18S rRNA genes were directly obtained from HiFi reads, which greatly surpassed the retrieval performance of NGS technology. We carried out a cross-sectional comparison among multiple assembly strategies, referred to as 'NGS', 'Hybrid (NGS+HiFi)', and 'HiFi'. Two MAGs and 29 viruses with circular genomes were reconstructed using HiFi reads alone, indicating the great power of the 'HiFi' approach to assemble high-quality microbial genomes. Among the 3 strategies, the 'Hybrid' approach produced the highest number of medium/high-quality MAGs and viral genomes, while the ratio of MAGs containing 16S rRNA genes was significantly improved in the 'HiFi' assembly results. Overall, our study provides a practical metagenomic resolution for analyzing complex environmental samples by taking advantage of both the short-read and HiFi long-read sequencing methods to extract the maximum amount of information, including data on prokaryotes, eukaryotes, and viruses, via the 'Hybrid' approach. IMPORTANCE To expand the understanding of microbial dark matter in the environment, we did the first comparative evaluation of multiple assembly strategies based on high-throughput short-read and HiFi data from lake sediments metagenomic sequencing. The results demonstrated great improvement of the 'Hybrid' assembly method (short-read next-generation sequencing data plus HiFi data) in the recovery of medium/high-quality MAGs and viral genomes. Further analysis showed that HiFi data is important to retrieve the complete circular prokaryotic and viral genomes. Meanwhile, hundreds of full-length 16S/18S rRNA genes were assembled directly from HiFi data, which facilitated the species composition studies of complex environmental samples, especially for understanding micro-eukaryotes. Therefore, the application of the latest HiFi long-read sequencing could greatly improve the metagenomic assembly integrity and promote environmental microbiome research.

Y-chromosome target enrichment reveals rapid expansion of haplogroup R1b-DF27 in Iberia during the Bronze Age transition

The Y chromosome can yield a unique perspective into the study of human demographic history. However, due to the repetitive nature of part of its sequence, only a small set of regions are suitable for variant calling and discovery from short-read sequencing data. These regions combined represent 8.9 Mbp or 0.14% of a diploid human genome. Consequently, investing in whole-genome sequencing to resolve Y-chromosome questions is poorly efficient. Here we use, as an alternative, target enrichment technology to greatly increase sequencing effectiveness, validating and applying the technique to 181 males, for 162 of whom we obtained a positive result. Additionally, 75 samples sequenced for the whole genome were also included, for a total sample size of 237. These samples were chosen for their Y chromosome haplogroup: R1b-DF27. In the context of European populations, and particularly in Iberia, this haplogroup stands out for its high frequency and its demographic history. Current evidence indicates that the diffusion of this haplogroup is related to the population movements that mark the cultural Bronze Age transition, making it remarkably interesting for population geneticists. The results of this study show the effects of the rapid radiation of the haplogroup in Spain, as even with the higher discriminating power of whole sequences, most haplotypes still fall within the R1b-DF27* paragroup rather than in the main derived branches. However, we were able to refine the ISOGG 2019–2020 phylogeny, and its two main subbranches, namely L176.2 and Z272, which present geographical differentiation between the Atlantic and Mediterranean coasts of Iberia.

Inferring Protein-DNA Binding Profiles at Interspersed Repeats Using HiChIP and PAtChER.

Alignment of short-read sequencing data to interspersed genomic repeats, such as transposable elements, can be problematic. This is especially true for evolutionarily young elements, which have not sufficiently diverged from each other to produce distinct and uniquely mappable reads. Mapping difficulties pose a challenge for studying the portfolio of epigenetic modifications and other chromatin regulators that bind to transposons and dictate their activity, which are typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Since ChIP-seq requires chromatin fragmentation to achieve appropriate resolution, longer reads do not appreciably improve mappability. Here, we present an experimental and computational protocol that couples ChIP-seq with 3D genome folding information to produce protein binding profiles with dramatically increased coverage at interspersed repeats.

A Pangenome Approach to Detect and Genotype TE Insertion Polymorphisms.

Pangenome graphs are flexible data structures that contain the genetic variation that exists in a population of genomes and describe the sequences of the many possible ensuing haplotypes. Here, we use such a pangenome graph to represent and genotype transposable element (TE) polymorphisms. By combining the transposable element annotation (Alus, L1s, and SVAs) of the human genome reference with novel transposable element insertions observed in two high-quality assemblies (HG002 and HG00733), we show how to create a transposable element pangenome that consists of ~1.2 million reference and 2939 non-reference transposable elements. We then demonstrate this approach by aligning short-read sequencing data and genotyping transposable element deletions and insertions with reasonable specificity and sensitivity (0.85F1-score).

Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum

BackgroundThe ability of malaria rapid diagnostic tests (RDTs) to effectively detect active infections is being compromised by the presence of malaria strains with genomic deletions at the hrp2 and hrp3 loci, encoding the antigens most commonly targeted in diagnostics for Plasmodium falciparum detection. The presence of such deletions can be determined in publically available P. falciparum whole genome sequencing (WGS) datasets. A computational approach was developed and validated, termed Gene Coverage Count and Classification (GC3), to analyse genome-wide sequence coverage data and provide informative outputs to assess presence and coverage profile of a target locus in WGS data. GC3 was applied to detect deletions at hrp2 and hrp3 (hrp2/3) and flanking genes in different geographic regions and across time points.MethodsGC3 uses Python and R scripts to extract locus read coverage metrics from mapped WGS data according to user-defined parameters and generates relevant tables and figures. GC3 was tested using WGS data for laboratory reference strains with known hrp2/3 genotypes, and its results compared to those of a hrp2/3-specific qPCR assay. Samples with at least 25% of coding region positions with zero coverage were classified as having a deletion. Publicly available sequence data was analysed and compared with published deletion frequency estimates.ResultsGC3 results matched the expected coverage of known laboratory reference strains. Agreement between GC3 and a hrp2/3-specific qPCR assay reported for 19/19 (100%) hrp2 deletions and 18/19 (94.7%) hrp3 deletions. Among Cambodian (n = 127) and Brazilian (n = 20) WGS datasets, which had not been previously analysed for hrp2/3 deletions, GC3 identified hrp2 deletions in three and four samples, and hrp3 deletions in 10 and 15 samples, respectively. Plots of hrp2/3 coding regions, grouped by year of sample collection, showed a decrease in median standardized coverage among Malawian samples (n = 150) suggesting the importance of a careful, properly controlled follow up to determine if an increase in frequency of deletions has occurred between 2007–2008 and 2014–2015. Among Malian (n = 90) samples, median standardized coverage was lower in 2002 than 2010, indicating widespread deletions present at the gene locus in 2002.ConclusionsThe GC3 tool accurately classified hrp2/3 deletions and provided informative tables and figures to analyse targeted gene coverage. GC3 is an appropriate tool when performing preliminary and exploratory assessment of locus coverage data.

NgsJulia: population genetic analysis of next-generation DNA sequencing data with Julia language.

A sound analysis of DNA sequencing data is important to extract meaningful information and infer quantities of interest. Sequencing and mapping errors coupled with low and variable coverage hamper the identification of genotypes and variants and the estimation of population genetic parameters. Methods and implementations to estimate population genetic parameters from sequencing data available nowadays either are suitable for the analysis of genomes from model organisms only, require moderate sequencing coverage, or are not easily adaptable to specific applications. To address these issues, we introduce ngsJulia, a collection of templates and functions in Julia language to process short-read sequencing data for population genetic analysis. We further describe two implementations, ngsPool and ngsPloidy, for the analysis of pooled sequencing data and polyploid genomes, respectively. Through simulations, we illustrate the performance of estimating various population genetic parameters using these implementations, using both established and novel statistical methods. These results inform on optimal experimental design and demonstrate the applicability of methods in ngsJulia to estimate parameters of interest even from low coverage sequencing data. ngsJulia provide users with a flexible and efficient framework for ad hoc analysis of sequencing data.ngsJulia is available from: https://github.com/mfumagalli/ngsJulia.

Abundant fungi dominate the complexity of microbial networks in soil of contaminated site: High-precision community analysis by full-length sequencing

During the past decade, the characterization of microbial community in soil of contaminated sites was primarily done by high-throughput short-read amplicon sequencing. However, due to the similarity of 16S rRNA and ITS genes amplicon sequences, the short-read approach often limits the microbial composition analysis at the species level. Here, we simultaneously performed full-length and short-read amplicon sequencing to clarify the community composition and ecological status of different microbial taxa in contaminated soil from a high-resolution perspective. We found that (1) full-length 16S rRNA gene sequencing gave better resolution for bacterial identification at all levels, while there were no significant differences between the two sequencing platforms for fungal identification in some samples. (2) Abundant taxa were vital for microbial co-occurrences network constructed by both full-length and short-read sequencing data, and abundant fungal species such as Mortierella alpine, Fusarium solani, Mrakia frigida, and Chaetomium homopilatum served as the keystone species. (3) Heavy metal correlated with the microbial community significantly, and bacterial community and its abundant taxa were assembled by deterministic process, while the other taxa were dominated by stochastic process. These findings contribute to the understanding of the ecological mechanisms and microbial interactions in site soil ecosystems and demonstrate that full-length sequencing has the potential to provide more details of microbial community.

Detection and Characterization of a De Novo Alu Retrotransposition Event Causing NKX2-1-Related Disorder.

Heterozygous NKX2-1 loss-of-function variants cause combinations of hyperkinetic movement disorders (MDs, particularly childhood-onset chorea), pulmonary dysfunction, and hypothyroidism. Mobile element insertions (MEIs) are potential disease-causing structural variants whose detection in routine diagnostics remains challenging. To establish the molecular diagnosis of two first-degree relatives with clinically suspected NKX2-1-related disorder who had negative NKX2-1 Sanger (SS), whole-exome (WES), and whole-genome (WGS) sequencing. The proband's WES was analyzed for MEIs. A candidate MEI in NKX2-1 underwent optimized SS after plasmid cloning. Functional studies exploring NKX2-1 haploinsufficiency at RNA and protein levels were performed. A 347-bp AluYa5 insertion with a 65-bp poly-A tail followed by a 16-bp duplication of the pre-insertion wild-type sequence in exon 3 of NKX2-1 (ENST00000354822.7:c.556_557insAlu541_556dup) segregated with the disease phenotype. We identified a de novo exonic AluYa5 insertion causing NKX2-1-related disorder in SS/WES/WGS-negative cases, suggesting that MEI analysis of short-read sequencing data or targeted long-read sequencing could unmask the molecular diagnosis of unsolved MD cases. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Personalized genome assembly for accurate cancer somatic mutation discovery using tumor-normal paired reference samples

BackgroundThe use of a personalized haplotype-specific genome assembly, rather than an unrelated, mosaic genome like GRCh38, as a reference for detecting the full spectrum of somatic events from cancers has long been advocated but has never been explored in tumor-normal paired samples. Here, we provide the first demonstrated use of de novo assembled personalized genome as a reference for cancer mutation detection and quantifying the effects of the reference genomes on the accuracy of somatic mutation detection.ResultsWe generate de novo assemblies of the first tumor-normal paired genomes, both nuclear and mitochondrial, derived from the same individual with triple negative breast cancer. The personalized genome was chromosomal scale, haplotype phased, and annotated. We demonstrate that it provides individual specific haplotypes for complex regions and medically relevant genes. We illustrate that the personalized genome reference not only improves read alignments for both short-read and long-read sequencing data but also ameliorates the detection accuracy of somatic SNVs and SVs. We identify the equivalent somatic mutation calls between two genome references and uncover novel somatic mutations only when personalized genome assembly is used as a reference.ConclusionsOur findings demonstrate that use of a personalized genome with individual-specific haplotypes is essential for accurate detection of the full spectrum of somatic mutations in the paired tumor-normal samples. The unique resource and methodology established in this study will be beneficial to the development of precision oncology medicine not only for breast cancer, but also for other cancers.