Short-read Whole-genome Sequencing Research Articles

RFC1 Repeat Distribution in the Cypriot Population: Study of a Large Cohort of Patients With Undiagnosed Ataxia and Non-Disease Controls.

The intronic biallelic AAGGG expansion in the replication factor C subunit 1 (RFC1) gene was recently associated with a phenotype combining cerebellar ataxia, neuropathy, and vestibular areflexia syndrome, as well as with late-onset ataxia. Following this discovery, studies in multiple populations extended the phenotypic and genotypic spectrum of this locus. Multiple benign and additional pathogenic configurations are currently known. Our main objectives were to study the prevalence of the pathogenic AAGGG expansion in the Cypriot population, to further characterize the RFC1 repeat locus allele distribution, and to search for possible novel repeat configurations. Cypriot undiagnosed patients, in the majority presenting at least with cerebellar ataxia and non-neurologic disease controls, were included in this study. A combination of conventional methods was used, including standard PCR flanking the repeat region, repeat-primed PCR, long-range PCR, and Sanger sequencing. Bioinformatics analysis of already available in-house short-read whole-genome sequencing data was also performed. A large group of undiagnosed patients (n = 194), mainly presenting with pure ataxia or with ataxia accompanied by neuropathy or additional symptoms, as well as a group of non-disease controls (n = 100), were investigated in the current study. Our findings include the diagnosis of 10 patients homozygous for the pathogenic AAGGG expansion and a high percentage of heterozygous AAGGG carriers in both groups. The benign AAAAGn, AAAGGn, and AAGAGn configurations were also identified in our cohorts. We also report and discuss the identification of 2 recently reported novel and possibly benign repeat configurations, AAAGGGn and AAGACn, thus confirming their existence in another distinct population, and we highlight an increased frequency of the AAAGGGn in the patient group, including a single case of homozygosity. Our findings indicate the existence of genetic heterogeneity regarding the RFC1 repeat configurations and that the AAGGG pathogenic expansion is a frequent cause of ataxia in the Cypriot population.

ACMGA: a reference-free multiple-genome alignment pipeline for plant species

BackgroundThe short-read whole-genome sequencing (WGS) approach has been widely applied to investigate the genomic variation in the natural populations of many plant species. With the rapid advancements in long-read sequencing and genome assembly technologies, high-quality genome sequences are available for a group of varieties for many plant species. These genome sequences are expected to help researchers comprehensively investigate any type of genomic variants that are missed by the WGS technology. However, multiple genome alignment (MGA) tools designed by the human genome research community might be unsuitable for plant genomes.ResultsTo fill this gap, we developed the AnchorWave-Cactus Multiple Genome Alignment (ACMGA) pipeline, which improved the alignment of repeat elements and could identify long (> 50 bp) deletions or insertions (INDELs). We conducted MGA using ACMGA and Cactus for 8 Arabidopsis (Arabidopsis thaliana) and 26 Maize (Zea mays) de novo assembled genome sequences and compared them with the previously published short-read variant calling results. MGA identified more single nucleotide variants (SNVs) and long INDELs than did previously published WGS variant callings. Additionally, ACMGA detected significantly more SNVs and long INDELs in repetitive regions and the whole genome than did Cactus. Compared with the results of Cactus, the results of ACMGA were more similar to the previously published variants called using short-read. These two MGA pipelines identified numerous multi-allelic variants that were missed by the WGS variant calling pipeline.ConclusionsAligning denovo assembled genome sequences could identify more SNVs and INDELs than mapping short-read. ACMGA combines the advantages of AnchorWave and Cactus and offers a practical solution for plant MGA by integrating global alignment, a 2-piece-affine-gap cost strategy, and the progressive MGA algorithm.

Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance

BackgroundKlebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019–February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples.ResultsThe draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins.ConclusionsThis study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

A novel framework for human leukocyte antigen (HLA) genotyping using probe capture-based targeted next-generation sequencing and computational analysis

Human leukocyte antigen (HLA) genes play pivotal roles in numerous immunological applications. Given the immense number of polymorphisms, achieving accurate high-throughput HLA typing remains challenging. This study aimed to harness the human pan-genome reference consortium (HPRC) resources as a potential benchmark for HLA reference materials. We meticulously annotated specific four field-resolution alleles for 11 HLA genes (HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5) from 44 high-quality HPRC personal genome assemblies. For sequencing, we crafted HLA-specific probes and conducted capture-based targeted sequencing of the genomic DNA of the HPRC cohort, ensuring focused and comprehensive coverage of the HLA region of interest. We used publicly available short-read whole-genome sequencing (WGS) data from identical samples to offer a comparative perspective. To decipher the vast amount of sequencing data, we employed seven distinct software tools: OptiType, HLA-VBseq, HISAT genotype, SpecHLA, T1K, QzType, and DRAGEN. Each tool offers unique capabilities and algorithms for HLA genotyping, allowing comprehensive analysis and validation of the results. We then compared these results with benchmarks derived from personal genome assemblies. Our findings present a comprehensive four-field-resolution HLA allele annotation for 44 HPRC samples. Significantly, our innovative targeted next-generation sequencing (NGS) approach for HLA genes showed superior accuracy compared with conventional short-read WGS. An integrated analysis involving QzType, T1K, and DRAGEN was developed, achieving 100% accuracy for all 11 HLA genes. In conclusion, our study highlighted the combination of targeted short-read sequencing and astute computational analysis as a robust approach for HLA genotyping. Furthermore, the HPRC cohort has emerged as a valuable assembly-based reference in this realm.

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

Landscape of genomic structural variations in Indian population-based cohorts: Deeper insights into their prevalence and clinical relevance

Structural variations (SV) are large (> 50 bp) genomic rearrangements comprising deletions, duplications, insertions, inversions, and translocations. Studying SVs is important because they play active and critical roles in regulating gene expression, determining disease predispositions, and identifying population-specific differences among individuals of diverse ancestries. However, SV discoveries in the Indian population using whole-genome sequencing (WGS) has been limited. In this study, using short-read WGS having an average 42X depth of coverage, we identify and characterize 36,210 SVs from 529 individuals enrolled in population-based cohorts in India. These SVs include 24,574 deletions, 2913 duplications, 8710 insertions, and 13 inversions. 1.26% (456 out of 36,210) of the identified SVs can potentially impact the coding regions of genes. Furthermore, 56 of these SVs are highly intolerant to loss-of-function changes to the mapped genes, and five SVs impacting ADAMTS17, CCDC40, and RHCE are common in our study individuals. Seven rare SVs significantly impact dosage sensitivity of genes known to be associated with various clinical phenotypes. Most of the SVs in our study are rare and heterozygous. This fine-scale SV discovery in the under-represented Indian population provides valuable insights that extends beyond Euro-centric human genetic studies.

Abstract 6247: Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) and linitis-plastica type gastric cancer (STAD) are the most representative refractory cancers even in the era of molecularly targeted therapy and immunotherapy. Extensive characterization of these tumors, especially regarding the genomic and epigenomic characteristics of pure cancer cells, has been hampered by the high stromal content in the primary tumor samples. Here we took advantage of cancer cells purified from malignant ascites and established corresponding cell lines to overcome the limitations associated with the analysis and to discover their vulnerabilities. Methods: We developed cell lines from malignant ascitic fluid samples obtained from 60 patients with PDAC and 59 patients with linitis-plastica type STAD. We performed comprehensive multi-omic analyses of these cell lines using long-read whole-genome sequencing (WGS) for complex somatic genomic alterations and for methylation aberration. We also conducted long-read RNA sequencing (iso-seq) for full-length isoform exploration, and chromatin immunoprecipitation coupled with sequencing (ChIP-seq) for enhancer analysis. Complementary short-read whole-genome sequencing and RNA sequencing were also performed. Results: Comprehensive transcriptome analyses identified a cluster with high epithelial-mesenchymal transition (EMT) characteristics, consisting of the majority of the PDAC cell lines and a half of the STAD cell lines. ChIP-seq analysis against H3K27ac revealed the distinct transcriptional circuit that generated EMT cluster-specific super-enhancers governed by SMAD3, independent of cancer type. Most of the transcriptional activity was controlled by the gene methylation. We also identified the differential expression levels of the splicing factors between the EMT-activated and the non-activated groups. Comprehensive isoform analysis by iso-seq revealed that the isoform usage was completely different between the EMT group and the other, especially in genes of the actomyosin pathway which confer characteristic features of the EMT cluster in the cell morphology and cell motility. Using the CHM13 reference genome, long-read WGS analyses revealed the recurrent structural alterations specific to the EMT group such as those involving CDKN2A. We also identified PDAC-specific structural alterations. Conclusion: We identified the distinct molecular characteristics of the EMT-activated group in PDAC and linitis-plastica type STAD which may confer the therapeutic resistance. Our findings provide an interesting insight for the further innovation of the treatment strategy for these intractable cancers. Citation Format: Yosuke Tanaka, Hiroyuki Mano. Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6247.

On the distribution and diversity of tissue-specific somatic mutations in honey bee (Apis mellifera) drones

Somatic mutations originate from both exogenous (e.g. UV radiation, chemical agents) and endogenous (e.g., DNA replication, defective DNA repair) sources and can have significant impacts on an animal’s reproductive success. This may be especially true for haploid organisms that are susceptible to any deleterious alleles inherited from their parent and any that arise over their lifetime. Unfortunately, little is known about the rate of somatic mutation accumulation across individuals and tissues of haplodiploid animal populations, the functional processes through which they arise, and their distribution across tissues and the genome. Here, we generated short-read whole-genome sequencing data for four tissues of haploid honey bee males. We paired this with estimates of telomere length and tissue-specific DNA content to address three major questions: is there variance in somatic mutational load across haploid individuals and specific tissues therein, does increased DNA content in a tissue contribute to somatic mutational load, and does telomere length correlate with mutational load? Our results suggest that variance in somatic mutational load is better captured across individuals than across tissues, that tissue-specific DNA content is not associated with somatic mutation load, and that variance in telomere length does not correlate with somatic mutation loads across tissues. To our knowledge, this is the first observational study on somatic mutational load in Apoidea and likely Hymenoptera. It serves as a useful advent for additional studies understanding the processes through which haploids tolerate or repair somatic mutations.

HAT: de novo variant calling for highly accurate short-read and long-read sequencing data.

de novo variants (DNVs) are variants that are present in offspring but not in their parents. DNVs are both important for examining mutation rates as well as in the identification of disease-related variation. While efforts have been made to call DNVs, calling of DNVs is still challenging from parent-child sequenced trio data. We developed Hare And Tortoise (HAT) as an automated DNV detection workflow for highly accurate short-read and long-read sequencing data. Reliable detection of DNVs is important for human genomics and HAT addresses this need. HAT is a computational workflow that begins with aligned read data (i.e. CRAM or BAM) from a parent-child sequenced trio and outputs DNVs. HAT detects high-quality DNVs from Illumina short-read whole-exome sequencing, Illumina short-read whole-genome sequencing, and highly accurate PacBio HiFi long-read whole-genome sequencing data. The quality of these DNVs is high based on a series of quality metrics including number of DNVs per individual, percent of DNVs at CpG sites, and percent of DNVs phased to the paternal chromosome of origin. https://github.com/TNTurnerLab/HAT.

Novel characterization of Alzheimer’s disease genetic loci using long‐read sequencing

AbstractBackgroundAlzheimer’s disease (AD) is up to 60‐80% heritable, but less than ∼20% is explained by studies analyzing single nucleotide variants (SNVs). One limitation of short‐read whole‐genome sequencing (SRS) is the standard read length of 150 base pairs, which does not enable the detection of longer structural variants (SVs). Here we sequenced individuals using long‐read sequencing (LRS), which can sequence reads with an average length of ∼20 kilobases, allowing us to identify SVs previously uncaptured.MethodAll participants underwent whole‐genome LRS (∼15x coverage) and a subgroup (84%) underwent SRS. Out of 576 participants (47% males, age = 70.6±7.9 y.o.), 115 were diagnosed with AD or mild cognitive impairment, 365 were healthy controls, and 96 were diagnosed with a synucleinopathy (either Parkinson or Lewy Body disease). Eighty‐three index SNVs from loci associated with AD risk through GWAS (Bellenguez et al., Nature Genetics 2022) were genotyped with 30x coverage SRS, in addition to APOE2‐4. A 1Mbp window was defined around these SNVs to construct the discovery range for SVs (Sniffles2 population mode). Linkage disequilibrium (LD) was assessed between SNVs and SVs (CubeX).ResultA total of 14854 SVs were found across the AD risk loci (Figure1). After LD calculation, N = 197 SVs had a R2>0.1 (Figure2). The SVs with the highest LD (R2>0.7) are reported in Table1, among which there is a 322 bp deletion in the 3’ UTR region of the TMEM106B in high LD (R2 = 0.918) with the intronic SNV rs13237518. This TMEM106B locus has been previously associated with the risk of frontotemporal lobar dementia with TDP‐43 pathology in addition to AD, but the causative SNV has not been identified yet. This large deletion may mediate TMEM106B’s risk‐modulating role in AD and FTLD‐TDP (Chemparathy et al. medRxiv 2023). At the complex MAPT locus, three SVs showed a high LD with rs199515.ConclusionInsights into the role of SVs in neurodegenerative disorders have been hampered due to limitations with SRS. Using LRS in a large AD‐related sample, we characterized for the first time the genetic variation of SVs in known AD risk loci and provide a roadmap to identify potential causal SVs driving the AD association signal.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones >5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

Cost-Effective Cas9-Mediated Targeted Sequencing of Spinocerebellar Ataxia Repeat Expansions

Hereditary repeat diseases are caused by an abnormal expansion of short tandem repeats in the genome. Among them, spinocerebellar ataxia (SCA) is a heterogeneous disease, and currently, 16 responsible repeats are known. Genetic diagnosis is obtained by analyzing the number of repeats through separate testing of each repeat. Although simultaneous detection of candidate repeats using current massively parallel sequencing technologies has been developed to avoid complicated multiple experiments, these methods are generally expensive. This study developed a cost-effective SCA repeat panel [Flongle SCA repeat panel sequencing (FLO-SCAp)] using Cas9-mediated targeted long-read sequencing and the smallest long-read sequencing apparatus, Flongle. This panel enabled the detection of repeat copy number changes, internal repeat sequences, and DNA methylation in seven patients with different repeat expansion diseases. The median (interquartile range) values of coverage and on-target rate were 39.5 (12 to 72) and 11.6% (7.5% to 16.5%), respectively. This approach was validated by comparing repeat copy number changes measured by FLO-SCAp and short-read whole-genome sequencing. A high correlation was observed between FLO-SCAp and short-read whole-genome sequencing when the repeat length was ≤250 bp (r=0.98; P<0.001). Thus, FLO-SCAp represents the most cost-effective method for conducting multiplex testing of repeats and can serve as the first-line diagnostic tool for SCA.

Population structure of Salmonella enterica Typhi in Harare, Zimbabwe (2012–19) before typhoid conjugate vaccine roll-out: a genomic epidemiology study

The continued emergence of Salmonella enterica serovar Typhi, with ever increasing antimicrobial resistance, necessitates the use of vaccines in endemic countries. A typhoid fever outbreak in Harare, Zimbabwe, in 2018 from a multidrug resistant S Typhi with additional resistance to ciprofloxacin was the catalyst for the introduction of a typhoid conjugate vaccine programme. We aimed to investigate the emergence and evolution of antimicrobial resistance of endemic S Typhi in Zimbabwe and to determine the population structure, gene flux, and sequence polymorphisms of strains isolated before a typhoid conjugate vaccine programme to provide a baseline for future evaluation of the effect of the vaccination programme. In this genomic epidemiology study, we used short-read whole-genome sequencing of S Typhi isolated from clinical cases of typhoid fever in Harare, Zimbabwe, between Jan 1, 2012, and Feb 9, 2019, to determine the S Typhi population structure, gene flux, and sequence polymorphisms and reconstructed the evolution of antimicrobial resistance. Maximum likelihood time-scaled phylogenetic trees of Zimbabwe isolates in the context of global isolates obtained from the National Center for Biotechnology Information were constructed to infer spread and emergence of antimicrobial resistance. The population structure of S Typhi in Harare, Zimbabwe, from 2012 to 2019 was dominated by multidrug resistant genotype 4.3.1.1.EA1 (H58) that spread to Zimbabwe from neighbouring countries in around 2009 (95% credible interval 2008·5-2010·0). Acquisition of an IncN plasmid carrying antimicrobial resistance genes including a qnrS gene and a mutation in the quinolone resistance determining region of gyrA gene contributed to non-susceptibility and resistance to quinolone antibiotics. A minority population of antimicrobial susceptible S Typhi genotype 3.3.1 strains were present throughout. The currently dominant S Typhi population is genotype 4.3.1.1 that spread to Zimbabwe and acquired additional antimicrobial resistance though acquisition of a plasmid and mutation in the gyrA gene. This study provides a baseline population structure for future evaluation of the effect of the typhoid conjugate vaccine programme in Harare. Bill & Melinda Gates Foundation and the Biotechnology and Biological Sciences Research Council Institute Strategic Programme.

Robust assembly of the aldehyde dehydrogenase Ald4p in Saccharomyces cerevisiae.

As part of our studies of yeast aldehyde dehydrogenase (Ald4p) assembly, we identified a population of transformants (SWORD strain) that show more robust filament formation of GFP-tagged Ald4p (Ald4p-GFP) than that of a wild type ALD4::GFP strain. Sequencing of the ALD4 gene in the SWORD strain showed that the increased assembly was not due to changes to the ALD4 coding sequence, suggesting that a second mutation site was altering Ald4p assembly. Using short-read whole-genome sequencing, we identified spontaneous mutations in FLO9. Introduction of the SWORD allele of FLO9 into a wild-type ALD4::GFP yeast strain revealed that the changes to FLO9 were a contributor to the increased length of Ald4p-GFP filaments we observe in the SWORD strain and that this effect was not due to an increase in Ald4p protein levels. However, the expression of the FLO9 (SWORD) allele in wild-type yeast did not fully recapitulate the length control defect we observed in SWORD strains, arguing that there are additional genes contributing to the filament length phenotype. For our future work, this FLO9 from SWORD will be tested whether it could show global effect, promoting the assembly of some other filament-forming enzymes.

MOM: A user-friendly Galaxy workflow to detect modifiers from genome sequencing data using C. elegans.

Genetic modifiers are variants modulating phenotypic outcomes of a primary detrimental variant. They contribute to rare diseases phenotypic variability, but their identification is challenging. Genetic screening with model organisms is a widely used method for demystifying genetic modifiers. Forward genetics screening followed by whole genome sequencing (WGS) allows the detection of variants throughout the genome, but typically produces thousands of candidate variants making the interpretation and prioritization process very time consuming and tedious. Despite WGS being more time and cost efficient, usage of computational pipelines specific to modifier identification remains a challenge for biological-experiment-focused laboratories doing research with model organisms. To facilitate a broader implementation of WGS in genetic screens, we have developed MOM, a Model Organism Modifier pipeline as a user-friendly Galaxy workflow. MOM analyses raw short-read WGS data and implements tailored filtering to provide a Candidate Variant List (CVL) short enough to be further manually curated. We provide a detailed tutorial to run the Galaxy workflow MOM and guidelines to manually curate the CVLs. We have tested MOM on published and validated C. elegans modifiers screening datasets. As WGS facilitates high-throughput identification of genetic modifiers in model organisms, MOM provides a user-friendly solution to implement the bioinformatics analysis of the short-read datasets in laboratories without expertise or support in Bioinformatics.

Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These datarevealeda distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocalpair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases recruited to the 100 000 Genomes Project

BackgroundCurrent clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm...

Surveillance of antimicrobial resistant Shiga toxin-producing E. coli O157:H7 in England, 2016-2020.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 are zoonotic pathogens and transmission to humans occurs via contaminated food or contact with infected animals. The aim of this study was to describe the frequency, and distribution across the phylogeny, of antimicrobial resistance (AMR) determinants in STEC O157:H7 isolated from human cases in England. Short-read whole-genome sequencing data from 1473 isolates of STEC O157:H7 from all seven sub-lineages (Ia-Ic, IIa-IIc and I/II) were mapped to genes known to confer phenotypic resistance to 10 different classes of antibiotic. Long-read sequencing was used to determine the location and genomic architecture of the AMR determinants within phylogenetic clusters exhibiting multidrug resistance. Overall, 216/1473 (14.7%) isolates had at least one AMR determinant, although the proportion of isolates exhibiting AMR varied by sub-lineage. The highest proportion of AMR determinants were detected in sub-lineages Ib (28/64, 43.7%), I/II (18/51, 35.3%) and IIc (122/440, 27.7%). In all sub-lineages, the most commonly detected AMR determinants conferred resistance to the aminoglycosides, tetracyclines and sulphonamides, while AMR determinants conferring resistance to fluroquinolones, macrolides and third-generation cephalosporins were rarely detected. Long-read sequencing analysis showed that the AMR determinants were co-located on the chromosome in sub-lineages Ib and lineage I/II, whereas those associated with sub-lineage IIc were encoded on the chromosome and/or large plasmids. AMR genes were unevenly distributed across the different sub-lineages of STEC O157:H7 and between different clades within the same sub-lineage. Long-read sequencing facilitates tracking the transmission of AMR at the pathogen and mobile genetic element level.

Genomic analysis of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli colonising adults in Blantyre, Malawi reveals previously undescribed diversity.

Escherichia coli is one of the most prevalent Gram-negative species associated with drug resistant infections. Strains that produce extended-spectrum beta-lactamases (ESBLs) or carbapenemases are both particularly problematic and disproportionately impact resource limited healthcare settings where last-line antimicrobials may not be available. A large number of E. coli genomes are now available and have allowed insights into pathogenesis and epidemiology of ESBL E. coli but genomes from sub-Saharan Africa (sSA) are significantly underrepresented. To reduce this gap, we investigated ESBL-producing E. coli colonising adults in Blantyre, Malawi to assess bacterial diversity and AMR determinants and to place these isolates in the context of the wider population structure. We performed short-read whole-genome sequencing of 473 colonising ESBL E. coli isolated from human stool and contextualised the genomes with a previously curated multi-country collection of 10 146 E. coli genomes and sequence type (ST)-specific collections for our three most commonly identified STs. These were the globally successful ST131, ST410 and ST167, and the dominant ESBL genes were bla CTX-M, mirroring global trends. However, 37 % of Malawian isolates did not cluster with any isolates in the curated multicountry collection and phylogenies were consistent with locally spreading monophyletic clades, including within the globally distributed, carbapenemase-associated B4/H24RxC ST410 lineage. A single ST2083 isolate in this collection harboured a carbapenemase gene. Long read sequencing demonstrated the presence of a globally distributed ST410-associated carbapenemase carrying plasmid in this isolate, which was absent from the ST410 strains in our collection. We conclude there is a risk that carbapenem resistance in E. coli could proliferate rapidly in Malawi under increasing selection pressure, and that both ongoing antimicrobial stewardship and genomic surveillance are critical as local carbapenem use increases.

Comparative Analysis of Structural Variant Callers on the Short-Read Whole-Genome Sequencing Data

In this study three structural variant callers (Manta, Smoove, Delly) were analysed on the whole-genome sequencing data using four different alignment algorithms: DRAGEN, GDC DNA-Seq Alignment Workflow, GDC DNA-Seq Alignment Workflow + GDC DNA-Seq Co-Cleaning Workflow, NovoAlign, different lengths of raw reads: 2 × 150 bp and 2 × 250 bp, different mean genome coverage values. Results were compared to etalon results of GIAB team. Structural variants validation was hold also with Sanger sequencing. Structural variants deletions and insertions as it turned out were best determined with Manta tool. We’ve got 89–96% of accuracy and 59–70% of sensitivity for analysed deletions, and also 96–99% of accuracy and 15–36% of sensitivity for insertions. Smoove and Delly showed less accurate and sensitive results (Smoove: 91–95% of accuracy and 8–54% of sensitivity for deletions, Delly: 78–87% of accuracy and 31–66% of sensitivity for deletions, 99–100% of accuracy and 1–13% of sensitivity for insertions). Simultaneous using of two or even three structural variant callers didn’t give a rise of accuracy and sensitivity for deletions. Analysis showed that accuracy and sensitivity of structural variant callers rise with the rising of mean genome coverage value, increasing of reads length from 150 to 250 bp influence in to varying degrees on the accuracy and sensitivity of individual tools. Another inference of this study was that accuracy of structural variants callers vary depends on structural variants size range. For example, Manta finds better deletions in the range from 200 and more bp, Delly – from 1000 to 10 000 bp, Smoove – from 200 to 10 000 bp.