Short-read Whole-genome Sequencing Research Articles

Unsuspected Clonal Spread of Methicillin-Resistant Staphylococcus aureus Causing Bloodstream Infections in Hospitalized Adults Detected Using Whole Genome Sequencing.

Though detection of transmission clusters of methicillin-resistant Staphylococcus aureus (MRSA) infections is a priority for infection control personnel in hospitals, the transmission dynamics of MRSA among hospitalized patients with bloodstream infections (BSIs) has not been thoroughly studied. Whole genome sequencing (WGS) of MRSA isolates for surveillance is valuable for detecting outbreaks in hospitals, but the bioinformatic approaches used are diverse and difficult to compare. We combined short-read WGS with genotypic, phenotypic, and epidemiological characteristics of 106 MRSA BSI isolates collected for routine microbiological diagnosis from inpatients in 2 hospitals over 12 months. Clinical data and hospitalization history were abstracted from electronic medical records. We compared 3 genome sequence alignment strategies to assess similarity in cluster ascertainment. We conducted logistic regression to measure the probability of predicting prior hospital overlap between clustered patient isolates by the genetic distance of their isolates. While the 3 alignment approaches detected similar results, they showed some variation. A gene family-based alignment pipeline was most consistent across MRSA clonal complexes. We identified 9 unique clusters of closely related BSI isolates. Most BSIs were healthcare associated and community onset. Our logistic model showed that with 13 single-nucleotide polymorphisms, the likelihood that any 2 patients in a cluster had overlapped in a hospital was 50%. Multiple clusters of closely related MRSA isolates can be identified using WGS among strains cultured from BSI in 2 hospitals. Genomic clustering of these infections suggests that transmission resulted from a mix of community spread and healthcare exposures long before BSI diagnosis.

Unravelling the tumour genome: The evolutionary and clinical impacts of structural variants in tumourigenesis.

Structural variants (SVs) represent a major source of aberration in tumour genomes. Given the diversity in the size and type of SVs present in tumours, the accurate detection and interpretation of SVs in tumours is challenging. New classes of complex structural events in tumours are discovered frequently, and the definitions of the genomic consequences of complex events are constantly being refined. Detailed analyses of short‐read whole‐genome sequencing (WGS) data from large tumour cohorts facilitate the interrogation of SVs at orders of magnitude greater scale and depth. However, the inherent technical limitations of short‐read WGS prevent us from accurately detecting and investigating the impact of all the SVs present in tumours. The expanded use of long‐read WGS will be critical for improving the accuracy of SV detection, and in fully resolving complex SV events, both of which are crucial for determining the impact of SVs on tumour progression and clinical outcome. Despite the present limitations, we demonstrate that SVs play an important role in tumourigenesis. In particular, SVs contribute significantly to late‐stage tumour development and to intratumoural heterogeneity. The evolutionary trajectories of SVs represent a window into the clonal dynamics in tumours, a comprehensive understanding of which will be vital for influencing patient outcomes in the future. Recent findings have highlighted many clinical applications of SVs in cancer, from early detection to biomarkers for treatment response and prognosis. As the methods to detect and interpret SVs improve, elucidating the full breadth of the complex SV landscape and determining how these events modulate tumour evolution will improve our understanding of cancer biology and our ability to capitalise on the utility of SVs in the clinical management of cancer patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.

BackgroundHypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes.MethodsNine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment.ResultsAmong the nine isolates, seven were carbapenem-resistant, two of which carried bla NDM-5 on an IncFII plasmid and five carried bla OXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB–IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, bla OXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition.ConclusionThe convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.

16p13.11p11.2 triplication syndrome: a new recognizable genomic disorder characterized by optical genome mapping and whole genome sequencing.

Highly identical segmental duplications (SDs) account for over 5% of the human genome and are enriched in the short arm of the chromosome 16. These SDs are susceptibility factors for recurrent chromosomal rearrangements mediated by non-allelic homologous recombination (NAHR). Chromosomal microarray analysis (CMA) has been widely used as the first-tier test for individuals with developmental disabilities and/or congenital anomalies and several genomic disorders involving the 16p-arm have been identified with this technique. However, the resolution of CMA and the limitations of short-reads whole genome sequencing (WGS) technology do not allow the full characterization of the most complex chromosomal rearrangements. Herein, we report on two unrelated patients with a de novo 16p13.11p11.2 triplication associated with a 16p11.2 duplication, detected by CMA. These patients share a similar phenotype including hypotonia, severe neurodevelopmental delay with profound speech impairment, hyperkinetic behavior, conductive hearing loss, and distinctive facial features. Short-reads WGS could not map precisely any of the rearrangement's breakpoints that lie within SDs. We used optical genome mapping (OGM) to determine the relative orientation of the triplicated and duplicated segments as well as the genomic positions of the breakpoints, allowing us to propose a mechanism involving recombination between allelic SDs and a NAHR event. In conclusion, we report a new clinically recognizable genomic disorder. In addition, the mechanism of these complex chromosomal rearrangements involving SDs could be unraveled by OGM.

Comparative Draft Genomes of Leishmania orientalis Isolate PCM2 (Formerly Named Leishmania siamensis) and Leishmania martiniquensis Isolate PCM3 from the Southern Province of Thailand.

Simple SummaryThis study successfully sequenced the draft genomes of the southern isolates of Leishmania orientalis and Leishmania martiniquensis in Thailand. The comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different emerging strains. Comparing the proteins of these southern strains to those of the northern ones and 14 other Leishmania species showed six protein groups with numerous unique proteins: 53 for the southern strain PCM2 of L. orientalis and 97 for the strain PCM3 of L. martiniquensis. Some of these proteins were related to virulence, drug resistance, drug target, and stress response, which could be targeted for further experimental characterization. Therefore, the findings could initiate further genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis) and Leishmania martiniquensis, has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.

Benchmarking the empirical accuracy of short-read sequencing across the M. tuberculosis genome.

Short-read whole-genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences and sequencing bias reduces the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias and GC content. Reference-based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results, we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems and more generally for WGS applications in other organisms. All relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation. Supplementary data are available at Bioinformatics online.

Comparative Whole-Genome Analysis of Russian Foodborne Multidrug-Resistant Salmonella Infantis Isolates.

Non-typhoidal Salmonella infections remain a significant public health problem worldwide. In this study, we present the first detailed genomic analysis report based on short-read (Illumina) whole-genome sequencing (WGS) of 45 multidrug-resistant (MDR) Salmonella enterica subsp. enterica serotype Infantis isolates from poultry and meat product samples obtained in Russia during 2018–2020, and long-read (MinION) WGS of five more representative isolates. We sought to determine whether foodborne S. Infantis have acquired new characteristics, traits, and dynamics in MDR growth in recent years. All sequenced isolates belonged to the sequence type ST32 and more than the half of isolates was characterized by six similar antimicrobial susceptibility profiles, most of which corresponded well with the antimicrobial resistance determinants to aminoglycosides, sulphonamides, tetracycline, and chloramphenicol revealed in silico. Some of the isolates were characterized by the presence of several types of plasmids simultaneously. Plasmid typing using WGS revealed Col440I, ColpVC, ColRNAI, IncFIB, IncFII, IncX1, IncHI2, IncHI2A, and IncN replicons. The identified virulence genes for 45 whole genomes of S. Infantis were similar and included 129 genes encoding structural components of the cell, factors responsible for successful invasion of the host, and secreted products. These data will be a valuable contribution to further comparative genomics of S. Infantis circulating in Russia, as well as to epidemiological surveillance of foodborne Salmonella isolates and investigations of Salmonella outbreaks.

Genomic analyses of the metastasis-derived prostate cancer cell lines LNCaP, VCaP, and PC3-AR.

The lymph node metastasis-derived LNCaP, the bone metastasis-derived PC3 (skull), and VCaP (vertebral) cell lines are widely used as preclinical models of human prostate cancer (CaP) and have been described in more than 19,000 publications. Here, we report on short-read whole-genome sequencing and genomic analyses of LNCaP, VCaP, and PC3 cells stably transduced with WT AR (PC3-AR). LNCaP, VCaP, and PC3-AR cell lines were sequenced to an average depth of more than 30-fold using Illumina short-read sequencing. Using various computational methods, we identified and compared the single-nucleotide variants, copy-number profiles, and the structural variants observed in the three cell lines. LNCaP cells are composed of multiple subpopulations, which results in nonintegral copy number states and a high mutational load when the data is analyzed in bulk. All three cell lines contain pathogenic mutations and homozygous deletions in genes involved in DNA mismatch repair, along with deleterious mutations in cell-cycle, Wnt signaling, and other critical cellular processes. PC3-AR cells have a truncating mutation in TP53 and do not express the p53 protein. The VCaP cells contain a homozygous gain-of-function mutation in TP53 (p.R248W) that promotes cancer invasion, metastasis, and progression and has also been observed in prostate adenocarcinomas. In addition, we detect the signatures of chromothripsis of the q arms of chromosome 5 in both PC3-AR and VCaP cells, strengthening the association of TP53 inactivation with chromothripsis reported in other systems. Our work provides a resource for genetic, genomic, and biological studies employing these commonly-used prostate cancer cell lines.

Long-read whole genome sequencing reveals HOXD13 alterations in synpolydactyly.

Synpolydactyly 1, also called syndactyly type II (SDTY2), is a genetic limb malformation characterized by polydactyly with syndactyly involving the webbing of the third and fourth fingers, and the fourth and fifth toes. It is caused by heterozygous alterations in HOXD13 with incomplete penetrance and phenotypic variability. In our study, a five-generation family with an SPD phenotype was enrolled in our Rare Disease Genomics Protocol. A comprehensive examination of three generations using Illumina short-read whole-genome sequencing (WGS) did not identify any causative variants. Subsequent WGS using Pacific Biosciences (PacBio) long-read HiFi Circular Consensus Sequencing (CCS) revealed a heterozygous 27-bp duplication in the polyalanine tract of HOXD13. Sanger sequencing of all available family members confirmed that the variant segregates with affected individuals. Reanalysis of an unrelated family with a similar SPD phenotype uncovered a 21-bp (7-alanine) duplication in the same region of HOXD13. Although ExpansionHunter identified these events in most individuals in a retrospective analysis, low sequence coverage due to high GC content in the HOXD13 polyalanine tract makes detection of these events challenging. Our findings highlight the value of long-read WGS in elucidating the molecular etiology of congenital limb malformation disorders.

Dissection of Highly Prevalent qnrS1-Carrying IncX Plasmid Types in Commensal Escherichia coli from German Food and Livestock.

Plasmids are mobile genetic elements, contributing to the spread of resistance determinants by horizontal gene transfer. Plasmid-mediated quinolone resistances (PMQRs) are important determinants able to decrease the antimicrobial susceptibility of bacteria against fluoroquinolones and quinolones. The PMQR gene qnrS1, especially, is broadly present in the livestock and food sector. Thus, it is of interest to understand the characteristics of plasmids able to carry and disseminate this determinant and therewith contribute to the resistance development against this class of high-priority, critically important antimicrobials. Therefore, we investigated all commensal Escherichia (E.) coli isolates, with reduced susceptibility to quinolones, recovered during the annual zoonosis monitoring 2017 in the pork and beef production chain in Germany (n = 2799). Through short-read whole-genome sequencing and bioinformatics analysis, the composition of the plasmids and factors involved in their occurrence were determined. We analysed the presence and structures of predominant plasmids carrying the PMQR qnrS1. This gene was most frequently located on IncX plasmids. Although the E. coli harbouring these IncX plasmids were highly diverse in their sequence types as well as their phenotypic resistance profiles, the IncX plasmids-carrying the qnrS1 gene were rather conserved. Thus, we only detected three distinct IncX plasmids carrying qnrS1 in the investigated isolates. The IncX plasmids were assigned either to IncX1 or to IncX3. All qnrS1-carrying IncX plasmids further harboured a β-lactamase gene (bla). In addition, all investigated IncX plasmids were transmissible. Overall, we found highly heterogenic E. coli harbouring conserved IncX plasmids as vehicles for the most prevalent qnr gene qnrS1. These IncX plasmids may play an important role in the dissemination of those two resistance determinants and their presence, transfer and co-selection properties require a deeper understanding for a thorough risk assessment.

Deciphering complex genome rearrangements in C. elegans using short-read whole genome sequencing

Genomic rearrangements cause congenital disorders, cancer, and complex diseases in human. Yet, they are still understudied in rare diseases because their detection is challenging, despite the advent of whole genome sequencing (WGS) technologies. Short-read (srWGS) and long-read WGS approaches are regularly compared, and the latter is commonly recommended in studies focusing on genomic rearrangements. However, srWGS is currently the most economical, accurate, and widely supported technology. In Caenorhabditis elegans (C. elegans), such variants, induced by various mutagenesis processes, have been used for decades to balance large genomic regions by preventing chromosomal crossover events and allowing the maintenance of lethal mutations. Interestingly, those chromosomal rearrangements have rarely been characterized on a molecular level. To evaluate the ability of srWGS to detect various types of complex genomic rearrangements, we sequenced three balancer strains using short-read Illumina technology. As we experimentally validated the breakpoints uncovered by srWGS, we showed that, by combining several types of analyses, srWGS enables the detection of a reciprocal translocation (eT1), a free duplication (sDp3), a large deletion (sC4), and chromoanagenesis events. Thus, applying srWGS to decipher real complex genomic rearrangements in model organisms may help designing efficient bioinformatics pipelines with systematic detection of complex rearrangements in human genomes.

Chromoanagenesis Event Underlies a de novo Pericentric and Multiple Paracentric Inversions in a Single Chromosome Causing Coffin-Siris Syndrome.

Chromoanagenesis is a descriptive term that encompasses classes of catastrophic mutagenic processes that generate localized and complex chromosome rearrangements in both somatic and germline genomes. Herein, we describe a 5-year-old female presenting with a constellation of clinical features consistent with a clinical diagnosis of Coffin–Siris syndrome 1 (CSS1). Initial G-banded karyotyping detected a 90-Mb pericentric and a 47-Mb paracentric inversion on a single chromosome. Subsequent analysis of short-read whole-genome sequencing data and genomic optical mapping revealed additional inversions, all clustered on chromosome 6, one of them disrupting ARID1B for which haploinsufficiency leads to the CSS1 disease trait (MIM:135900). The aggregate structural variant data show that the resolved, the resolved derivative chromosome architecture presents four de novo inversions, one pericentric and three paracentric, involving six breakpoint junctions in what appears to be a shuffling of genomic material on this chromosome. Each junction was resolved to nucleotide-level resolution with mutational signatures suggestive of non-homologous end joining. The disruption of the gene ARID1B is shown to occur between the fourth and fifth exon of the canonical transcript with subsequent qPCR studies confirming a decrease in ARID1B expression in the patient versus healthy controls. Deciphering the underlying genomic architecture of chromosomal rearrangements and complex structural variants may require multiple technologies and can be critical to elucidating the molecular etiology of a patient’s clinical phenotype or resolving unsolved Mendelian disease cases.

Exploring the missing heritability in subjects with hearing loss, enlarged vestibular aqueducts, and a single or no pathogenic SLC26A4 variant

Pathogenic variants in SLC26A4 have been associated with autosomal recessive hearing loss (arHL) and a unilateral or bilateral enlarged vestibular aqueduct (EVA). SLC26A4 is the second most frequently mutated gene in arHL. Despite the strong genotype–phenotype correlation, a significant part of cases remains genetically unresolved. In this study, we investigated a cohort of 28 Dutch index cases diagnosed with HL in combination with an EVA but without (M0) or with a single (M1) pathogenic variant in SLC26A4. To explore the missing heritability, we first determined the presence of the previously described EVA-associated haplotype (Caucasian EVA (CEVA)), characterized by 12 single nucleotide variants located upstream of SLC26A4. We found this haplotype and a delimited V1-CEVA haplotype to be significantly enriched in our M1 patient cohort (10/16 cases). The CEVA haplotype was also present in two M0 cases (2/12). Short- and long-read whole genome sequencing and optical genome mapping could not prioritize any of the variants present within the CEVA haplotype as the likely pathogenic defect. Short-read whole-genome sequencing of the six M1 cases without this haplotype and the two M0/CEVA cases only revealed previously overlooked or misinterpreted splice-altering SLC26A4 variants in two cases, who are now genetically explained. No deep-intronic or structural variants were identified in any of the M1 subjects. With this study, we have provided important insights that will pave the way for elucidating the missing heritability in M0 and M1 SLC26A4 cases. For pinpointing the pathogenic effect of the CEVA haplotype, additional analyses are required addressing defect(s) at the RNA, protein, or epigenetic level.

MRSA strains with distinct accessory genes predominate at different ages in cystic fibrosis.

Methicillin resistant Staphylococcus aureus (MRSA) is prevalent and consequential in cystic fibrosis (CF). Whole genome sequencing (WGS) could reveal genomic differences in MRSA associated with poorer outcomes or detect MRSA transmission. To identify MRSA genes associated with low lung function and potential MRSA transmission in CF. We collected 97 MRSA isolates from 74 individuals with CF from 2017 and performed short-read WGS. We determined sequence type (ST) and the phylogenetic relationship between isolates. We aligned accessory genes from 25 reference genomes to genome assemblies, classified isolates by accessory gene content, and correlated the accessory genome to clinical outcomes. The most prevalent ST were ST5 (N = 55), ST8 (N = 15), and ST105 (N = 14). Closely related MRSA strains were shared by family members with CF, but rarely between unrelated individuals. Three clusters of MRSA were identified by accessory genome content. Cluster A, including ST5 and ST105, was highly prevalent at all ages. Cluster B, including ST8, was more limited to younger patients. Cluster C included 6 distantly related strains. Patients 20 years old and younger infected with Cluster A had lower forced expiratory volume in the first second (FEV1 )and higher sputum biomass compared to similar-aged patients with Cluster B. In this CF cohort, we identified MRSA subtypes that predominate at different ages and differ by accessory gene content. The most prevalent cluster of MRSA, including ST5 and ST105, was associated with lower FEV1 . ST8 MRSA was more common in younger patients and thus has the potential to rise in prevalence as these patients age.

Description of novel resistance islands harbouring blaCTX-M-2 in IncC type 2 plasmids

We sequenced two IncA/C plasmids harbouring blaCTX-M-2 in Klebsiella pneumoniae clinical isolates and compared their antibiotic resistance islands. Transconjugants were obtained from two clinical K. pneumoniae isolates harbouring blaCTX-M-2. Plasmid DNA from transconjugants underwent short-read whole-genome sequencing, reads were assembled, and gaps were closed by PCR and sequencing. Determination of plasmid replicons, antibiotic resistance genes, identification and characterisation of insertion sequence (IS) elements, and comparison with publicly available plasmid sequences were performed. blaCTX-M-2 was located in a complex class 1 integron In35::ISCR1::blaCTX-M-2, inserted in two different transposons designated Tn7057 and Tn7058, that reside in the resistance islands of plasmids pUR-KP0923 and pUR-KP1025, respectively. The general modules of both transposons were In35::ISCR1::blaCTX-M-2-Tn1000-like-Tn2*-ISKpn11-12-13 variable module-ΔTn21. In Tn7057 there was ΔIS10R-catA2 associated with an additional ISKpn13. Both plasmids belonged to IncC type 2 and ST3. pUR-KP0923 was 167 138 bp in length and had a 37 926-bp resistance island at position 4 (RI-4). Plasmid pUR-KP1025 was 168 128 bp with a RI-4 of 36 222 bp. This report describes the molecular nature of two transposons (Tn7057 and Tn7058) harbouring blaCTX-M-2 that reside in IncC type 2 ST3 plasmids. These transposons mediate resistance to oxyimino-cephalosporins, gentamicin and, in the case of Tn7057, chloramphenicol. CTX-M-2 is an important extended-spectrum β-lactamase (ESBL) to South American epidemiology. It is remarkable that despite being only two plasmid sequences, the information revealed here could contribute to a better understanding of the resistance islands from IncC type 2 plasmids.

Transgene Mapping and Genotyping Protocol Design in a Transgenic Mouse Strain by Short‐Read Whole‐Genome Sequencing

[Aim] When creating transgenic mice, multiple lines with the same transgene are often created. For example, our laboratory has three lines of hAce2 transgenic mice (hAce2-Tg). For one of them (hAce2-Tg#16), we mapped the transgene using short-read whole-genome sequencing and designed genotyping PCR to identify the line. [Methods] We extracted genomic DNA from kidneys of hAce2-Tg#16. Using PCR-free libraries prepared with the genomic DNA, paired-end 150-bp reads were performed by DNBSEQG-400 sequencer (MGI Tech) (outsourced). In order to find the junction between the genome and the transgene, we created two search programs: one to search for matches between the transgene and each read and its complementary sequence from the front (5′ side, head-match), and the other to search for matches from the back (3′ side, tail-match). We selected reads with a match length of 5 to 145 bases. After the search, we selected reads containing the mouse genome sequence in the non-matching part by BLAST search of the Ensembl database. Based on this sequence information, we designed primers for genotyping PCR based on the flanking primer method. All animal experiments were conducted in accordance with the guidelines for animal experiments of the National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan (authorization number: DS22-55). [Results and Discussion] In a total of 882,577,032 reads (total read length: about 132 GBase), the total numbers of head-matching and tail-matching reads were 1,498 and 3,060, respectively. Among them, eight reads in the head-matching reads had the same genomic sequence. Its genomic location was in the intron of the Nrxn3 gene on chromosome 12. The transgene was truncated by 82 bases from the 3′ end. On the other hand, none of the tail-matching reads contained the neighboring genomic sequence. Genotyping PCRs with flanking primers we designed based on the sequence near the junction between transgene and genome sequences confirmed that the presence or absence of the transgene only in the line (hAce2-Tg#16). The authors have already reported examples of transgene mapping by Genomic Walking (Exp Anim 53: 103-111, 2004) and whole genome sequencing of long reads (Exp Anim 69: 279-286, 2020). This study confirmed that short-read whole-genome sequencing was also useful for mapping transgenes and designing PCR for genotyping of transgenic mice.

Genomic Characterization of the Barnacle Balanus improvisus Reveals Extreme Nucleotide Diversity in Coding Regions

Barnacles are key marine crustaceans in several habitats, and they constitute a common practical problem by causing biofouling on man-made marine constructions and ships. Despite causing considerable ecological and economic impacts, there is a surprising void of basic genomic knowledge, and a barnacle reference genome is lacking. We here set out to characterize the genome of the bay barnacle Balanus improvisus (= Amphibalanus improvisus) based on short-read whole-genome sequencing and experimental genome size estimation. We show both experimentally (DNA staining and flow cytometry) and computationally (k-mer analysis) that B. improvisus has a haploid genome size of ~ 740 Mbp. A pilot genome assembly rendered a total assembly size of ~ 600 Mbp and was highly fragmented with an N50 of only 2.2 kbp. Further assembly-based and assembly-free analyses revealed that the very limited assembly contiguity is due to the B. improvisus genome having an extremely high nucleotide diversity (π) in coding regions (average π ≈ 5% and average π in fourfold degenerate sites ≈ 20%), and an overall high repeat content (at least 40%). We also report on high variation in the α-octopamine receptor OctA (average π = 3.6%), which might increase the risk that barnacle populations evolve resistance toward antifouling agents. The genomic features described here can help in planning for a future high-quality reference genome, which is urgently needed to properly explore and understand proteins of interest in barnacle biology and marine biotechnology and for developing better antifouling strategies.

Expectations and blind spots for structural variation detection from long-read assemblies and short-read genome sequencing technologies

Virtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and medical genetic initiatives are reliant upon short-read whole-genome sequencing (srWGS), which presents challenges for the detection of structural variants (SVs) relative to emerging long-read WGS (lrWGS) technologies. Given this ubiquity of srWGS in large-scale genomics initiatives, we sought to establish expectations for routine SV detection from this data type by comparison with lrWGS assembly, as well as to quantify the genomic properties and added value of SVs uniquely accessible to each technology. Analyses from the Human Genome Structural Variation Consortium (HGSVC) of three families captured ~11,000 SVs per genome from srWGS and ~25,000 SVs per genome from lrWGS assembly. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplication (SD) and simple repeat (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of reference sequence, we observed extremely high (93.8%) concordance between technologies for deletions in these datasets. In contrast, lrWGS was superior for detection of insertions across all genomic contexts. Given that non-SD/SR sequences encompass 95.9% of currently annotated disease-associated exons, improved sensitivity from lrWGS to discover novel pathogenic deletions in these currently interpretable genomic regions is likely to be incremental. However, these analyses highlight the considerable added value of assembly-based lrWGS to create new catalogs of insertions and transposable elements, as well as disease-associated repeat expansions in genomic sequences that were previously recalcitrant to routine assessment.

Molecular characterization of clinical isolates of Enterobacterales with elevated MIC values for aztreonam-avibactam from the INFORM global surveillance study, 2012–2017

ObjectivesWhile aztreonam-avibactam is a potent β-lactam-β-lactamase-inhibitor combination, reduced in vitro activity against some Enterobacterales isolates has been reported. In this study, globally collected clinical isolates of Enterobacterales with elevated minimum inhibitory concentrations (MICs) for aztreonam-avibactam were examined for potential resistance mechanisms. MethodsIsolates with aztreonam-avibactam MICs ≥8 μg/mL (n = 55: Escherichia coli, n = 38; Enterobacter cloacae, n = 10; Klebsiella pneumoniae, n = 3; others, n = 4) and <8 μg/mL (n = 18) collected for the INFORM global surveillance programme were characterized by short read whole-genome sequencing. Sequences were inspected for the presence of β-lactamase genes, penicillin-binding protein (PBP) mutations, and disruptions in the coding sequences of porin genes. ResultsAll isolates of E. coli testing with aztreonam-avibactam MIC values ≥8 μg/mL carried a previously documented four-amino-acid insertion in PBP3 at position 333 of YRI(K/N/P). Such mutations were absent in isolates with MICs <2 μg/mL (n = 6). Among other species, carriage of PER- or VEB-type β-lactamases was identified in 10/17 (58.8%) of isolates testing with aztreonam-avibactam MICs ≥8 μg/mL, but no isolates with lower MIC values (n = 11). ConclusionsPBP3 mutations are known to confer resistance to aztreonam in E. coli, providing a rationale for the elevated MIC values for aztreonam-avibactam in these isolates. Elevated MICs in other isolates were associated with the carriage of PER-type β-lactamases, which have been previously shown to be inhibited less effectively by avibactam than other Class A β-lactamases and may contribute to this phenotype. Other resistance mechanisms contributing to poor in vitro activity for aztreonam-avibactam in some of these isolates are not yet elucidated.

A benchmark and an algorithm for detecting germline transposon insertions and measuring de novo transposon insertion frequencies.

Transposons are genomic parasites, and their new insertions can cause instability and spur the evolution of their host genomes. Rapid accumulation of short-read whole-genome sequencing data provides a great opportunity for studying new transposon insertions and their impacts on the host genome. Although many algorithms are available for detecting transposon insertions, the task remains challenging and existing tools are not designed for identifying de novo insertions. Here, we present a new benchmark fly dataset based on PacBio long-read sequencing and a new method TEMP2 for detecting germline insertions and measuring de novo ‘singleton’ insertion frequencies in eukaryotic genomes. TEMP2 achieves high sensitivity and precision for detecting germline insertions when compared with existing tools using both simulated data in fly and experimental data in fly and human. Furthermore, TEMP2 can accurately assess the frequencies of de novo transposon insertions even with high levels of chimeric reads in simulated datasets; such chimeric reads often occur during the construction of short-read sequencing libraries. By applying TEMP2 to published data on hybrid dysgenic flies inflicted by de-repressed P-elements, we confirmed the continuous new insertions of P-elements in dysgenic offspring before they regain piRNAs for P-element repression. TEMP2 is freely available at Github: https://github.com/weng-lab/TEMP2.