Whole-genome Single Nucleotide Polymorphism Research Articles

Landscape of meiotic crossovers in Hericium erinaceus

Meiotic crossover shows marked interspecific and intraspecific variation, and knowledge about the molecular mechanism of crossover variation remains limited. Herein, we described the genome-wide scanning of crossover in one mushroom-forming fungus Hericium erinaceus. Utilizing the whole-genome single-nucleotide polymorphism (SNP) data-sets of a 127 F1 haploid progeny, we localized a total of 1316 crossover events and found that they were more likely to occur in the genic than intergenic regions. More than 30 % of the crossovers were concentrated in 59 crossover hotspots that were preferentially located close to chromosome ends. We then examined the genomic features around crossover hotspots. Results showed that the crossover hotspots were associated with increased gene density and guanine-cytosine (GC) content. An 8-bp GC-rich motif (GCGTCAGC) was found to be significantly enriched in these hotspots. The presence of mating-type loci affected the crossover at local scale rather than the overall crossover number. In order to dissect the genetic mechanisms shaping crossover variation, we then conducted quantitative trait locus (QTL) mapping for the total crossovers (TCO) and the crossover events that solely occurred within hotspots (HCO). Genome-wide QTL scanning identified four TCO-QTLs and two HCO-QTLs, which all located within or next to the crossover-hotspots. Crossover variations were shaped by multiple small-effect loci, with individual QTL contributing 6.9 %–11.7 % of variation. A few recombination pathway genes, including Spo11, Msh5, and Smc5 were found to be co-localized with the mapped crossover QTLs. Taken together, findings of this study offer insights into the crossover distribution and genetic factors conferring crossover variation in H. erinaceus, and advance our understandings for meiotic recombination in mushroom-forming fungi.

Genotyping of Francisella tularensis subsp. holarctica from Hares in Germany.

Francisella tularensis is the causative agent of the zoonotic disease tularemia. In Germany, most human infections are caused by contact with infected hares. The aim of this study was to characterize Francisella tularensis subsp. holarctica strains isolated from hares in Germany and to develop bioinformatics tools to analyze their genetic relatedness. In total, 257 German isolates—obtained mainly from hares (n = 233), other vertebrate animals, and ticks, but also from humans (n = 3)—were analyzed within this study. Publically available sequence data from 49 isolates were used to put our isolates into an epidemiological context and to compare isolates from natural foci and humans. Whole-genome sequences were analyzed using core-genome Multi-Locus-Sequence-Typing, canonical Single Nucleotide Polymorphism (SNP) typing and whole-genome SNP typing. An overall conformity of genotype clustering between the typing methods was found, albeit with a lower resolution for canonical single SNP typing. The subclade distribution, both on local and national levels, among strains from humans and hares was similar, suggesting circulation of the same genotypes both in animals and humans. Whilst close to identical isolates of the same subclade were found distributed over large areas, small geographical foci often harbored members of different subclades. In conclusion, although genomic high-resolution typing was shown to be robust, reproducible and allowed the identification of highly closely related strains, genetic profiling alone is not always conclusive for epidemiological linkage of F. tularensis strains.

Definition of a genetic relatedness cutoff to exclude recent transmission of meticillin-resistant Staphylococcus aureus: a genomic epidemiology analysis

SummaryBackgroundWhole-genome sequencing (WGS) can be used in genomic epidemiology investigations to confirm or refute outbreaks of bacterial pathogens, and to support targeted and efficient infection control interventions. We aimed to define a genetic relatedness cutoff, quantified as a number of single-nucleotide polymorphisms (SNP), for meticillin-resistant Staphylococcus aureus (MRSA), above which recent (ie, within 6 months) patient-to-patient transmission could be ruled out.MethodsWe did a retrospective genomic and epidemiological analysis of MRSA data from two prospective observational cohort studies in the UK to establish SNP cutoffs for genetic relatedness, above which recent transmission was unlikely. We used three separate approaches to calculate these thresholds. First, we applied a linear mixed model to estimate the S aureus substitution rate and 95th percentile within-host diversity in a cohort in which multiple isolates were sequenced per individual. Second, we applied a simulated transmission model to this same genomic dataset. Finally, in a second cohort, we determined the genetic distance (ie, the number of SNPs) that would capture 95% of epidemiologically linked cases. We applied the three approaches to both whole-genome and core-genome sequences.FindingsIn the linear mixed model, the estimated substitution rate was roughly 5 whole-genome SNPs (wgSNPs) or 3 core-genome SNPs (cgSNPs) per genome per year, and the 95th percentile within-host diversity was 19 wgSNPs or 10 cgSNPs. The combined SNP cutoffs for detection of MRSA transmission within 6 months per this model were thus 24 wgSNPs or 13 cgSNPs. The simulated transmission model suggested that cutoffs of 17 wgSNPs or 12 cgSNPs would detect 95% of MRSA transmission events within the same timeframe. Finally, in the second cohort, cutoffs of 22 wgSNPs or 11 cgSNPs captured 95% of epidemiologically linked cases within 6 months.InterpretationOn the basis of our results, we propose conservative cutoffs of 25 wgSNPs or 15 cgSNPS above which transmission of MRSA within the previous 6 months can be ruled out. These cutoffs could potentially be used as part of a genomic sequencing approach to the management of outbreaks of MRSA in conjunction with traditional epidemiological techniques.FundingUK Department of Health, Wellcome Trust, UK National Institute for Health Research.

Detecting Genetic Ancestry and Adaptation in the Taiwanese Han People.

The Taiwanese people are composed of diverse indigenous populations and the Taiwanese Han. About 95% of the Taiwanese identify themselves as Taiwanese Han, but this may not be a homogeneous population because they migrated to the island from various regions of continental East Asia over a period of 400 years. Little is known about the underlying patterns of genetic ancestry, population admixture, and evolutionary adaptation in the Taiwanese Han people. Here, we analyzed the whole-genome single-nucleotide polymorphism genotyping data from 14,401 individuals of Taiwanese Han collected by the Taiwan Biobank and the whole-genome sequencing data for a subset of 772 people. We detected four major genetic ancestries with distinct geographic distributions (i.e., Northern, Southeastern, Japonic, and Island Southeast Asian ancestries) and signatures of population mixture contributing to the genomes of Taiwanese Han. We further scanned for signatures of positive natural selection that caused unusually long-range haplotypes and elevations of hitchhiked variants. As a result, we identified 16 candidate loci in which selection signals can be unambiguously localized at five single genes: CTNNA2, LRP1B, CSNK1G3, ASTN2, and NEO1. Statistical associations were examined in 16 metabolic-related traits to further elucidate the functional effects of each candidate gene. All five genes appear to have pleiotropic connections to various types of disease susceptibility and significant associations with at least one metabolic-related trait. Together, our results provide critical insights for understanding the evolutionary history and adaption of the Taiwanese Han population.

RNA-sequencing of IDH-wild-type glioblastoma with chromothripsis identifies novel gene fusions with potential oncogenic properties

Glioblastoma (GBM) is the most frequent and most aggressive form of glioma. It is characterized by marked genomic instability, which suggests that chromothripsis (CT) might be involved in GBM initiation. Recently, CT has emerged as an alternative mechanism of cancer development, involving massive chromosome rearrangements in a one-step catastrophic event. The aim of the study was to detect CT in GBM and identify novel gene fusions in CT regions. One hundred and seventy IDH-wild-type GBM were screened for CT patterns using whole-genome single nucleotide polymorphism (SNP) arrays. RNA sequencing was performed in 52 GBM with CT features to identify gene fusions within CT regions. Forty tumors (40/52, 77%) harbored at least one gene fusion within CT regions. We identified 120 candidate gene fusions, 30 of which with potential oncogenic activities. We validated 11 gene fusions, which involved the most recurrent fusion partners (EGFR, SEPT14, VOPP1 and CPM), by RT-PCR and Sanger sequencing. The occurrence of CT points to underlying gene fusions in IDH-wild-type GBM. CT provides exciting new research avenues in this highly aggressive cancer.

An Outbreak of USA300 Methicillin-Resistant Staphylococcus aureus Among People With HIV in Japan.

USA300 produces Panton-Valentin leucocidin (PVL) and is known as a predominant community-associated methicillin-resistant Staphylococcus aureus (MRSA) strain in the United States, but it was extremely rare in Japan. We report here an outbreak of USA300 in people with HIV (PWH) in Tokyo, Japan. We analyzed the cases of PVL-MRSA infection between 2010 and 2020 and screened for nasal colonization of PVL-MRSA in PWH who visited an HIV/AIDS referral hospital from December 2019 to March 2020. Whole-genome sequencing-based single nucleotide polymorphism (SNP) analysis was performed on these isolates. During the study period, a total of 21 PVL-MRSA infections in 14 patients were identified after 2014. The carriage prevalence was 4.3% (12/277) and PVL-MRSA carriers were more likely to have sexually transmitted infections (STIs) within a year compared with patients who had neither a history of PVL-MRSA infection nor colonization (33.3% [4/12] vs 10.1% [26/258]; P = .03). SNP analysis showed that all 26 isolates were ST8-SCCmecIVa-USA300. Twenty-four isolates were closely related (≤100 SNP differences) and had the nonsynonymous SNPs associated with carbohydrate metabolism and antimicrobial tolerance. An outbreak of USA300 has been occurring among PWH in Tokyo and a history of STI was a risk of colonization.

Genomic analysis of Clostridioides difficile in two regions reveals a diversity of strains and limited transmission

Background: The epidemic NAP1/027 Clostridioides difficile strain (MLST1, ST1) that emerged in the mid-2000 is on the decline. The current distribution of C. difficile strain types and their transmission dynamics are poorly defined. We performed whole-genome sequencing (WGS) of C. difficile isolates in 2 regions to identify the predominant multilocus sequence types (MLSTs) in community- and healthcare-associated cases and potential transmission between cases using whole-genome single-nucleotide polymorphism (SNP) analysis. Methods: Isolates were collected through the CDC Emerging Infections Program population-based surveillance for C. difficile infections (CDI) for 3 months between 2016 and 2017 in 5 Minnesota counties and 1 New York county. Isolates were limited to incident cases (CDI in a county resident with no positive C. difficile test in the preceding 8 weeks). Cases were classified as healthcare associated (HA-CDI) or community associated (CA-CDI) based on healthcare exposures as previously described. WGS was performed on an Illumina Miseq. The CFSAN (FDA) pipeline was used to compute whole-genome SNPs, SPAdes was used for assembly, and MLST was assigned according to www.pubmlst.org. Results: Of 431 isolates, 269 originated from New York and 162 from Minnesota; 203 cases were classified as CA-CDI and 221 as HA-CDI. The proportion of CA-CDI cases was higher in Minnesota than in New York: 62% vs 38%. The predominant MLSTs across both sites were ST42 (9%), ST8 (8%), and ST2 (8%). MLSTs more frequently encountered in HA-CDI than CA-CDI included ST1 (note that this ST includes PCR Ribotype 027; 76% HA-CDI), ST53 (84% HA-CDI), and ST43 (80% HA-CDI). In contrast, ST110 (63% CA-CDI) and ST3 (67% CA-CDI) were more commonly isolated from CA-CDI cases. ST1 accounted for 7.6% of circulating strains and was more common in New York than Minnesota (10% vs 3%) and was concentrated among New York HA-CDI cases. Also, 412 isolates (1 per patient) were included in the final whole-genome SNP analysis. Of these, only 12 pairs were separated by 0–3 SNPs, indicating potential transmission, and most involved HA-CDI cases. ST1, ST17, and ST46 accounted for 8 of 12 pairs, with ST17 and ST46 potentially forming small clusters. Conclusions: This analysis provides a snapshot of the current genomic epidemiology of C. difficile across 2 geographically and epidemiologically distinct regions of the United States and supports other studies suggesting that the role of direct transmission in the spread of CDI may be limited.Funding: NoneDisclosures: None

Abstract P125: Genes And Pathways Associated With Apparent Treatment-resistant Hypertension In Patients With Chronic Kidney Disease: An Analysis Of The Cric Study

Patients with chronic kidney disease (CKD) have a 4-fold increased risk of developing apparent treatment-resistant hypertension (aTRH) compared with the general population. This study aimed to identify key genes and pathways associated with aTRH based on data from the Chronic Renal Insufficiency Cohort (CRIC) study. In this study, 823,094 whole-genome single-nucleotide polymorphisms (SNPs) of 3,024 patients (1,215 with aTRH defined by 2018 American Heart Association Scientific Statements and 1,809 without aTRH) were used for the analysis. First, we identified 997 significant (p<0.05) genes associated with aTRH using the SNP-set kernel association test (SKAT) analysis after controlling for race, gender, body mass index, and diabetes. The top 5 significant genes identified were NMNAT3 (p=1.2х10-4, coding for an enzyme that catalyzes nicotinamide adenine dinucleotide synthesis), SLC2A13 (p=1.4х10-4, coding for a glucose transporter), PTBP1 (p=1.7х10-4, coding for an RNA-binding protein that controls alternative splicing), IZUMO4 (p=1.9х10-4, coding for a protein that facilitates fertilization), and CCDC9 (p=1.9х10-4, coding for coiled-coil domain-containing protein 9). Next, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses based on significant genes obtained from SKAT analysis. The identified top 5 GO pathways were “Metaphase plate congression” (p=1.3х10-4), “Histone deacetylase binding” (p=1.3х10-4), “Transcription regulatory region DNA binding” (p=1.7х10-4), “Cytokine-mediated signaling pathway” (p=1.8х10-4), and “Phosphotyrosine residue binding” (p=6.6х10-4). The top 5 KEGG pathways identified were “RNA degradation” (p=1.1х10-4), “Viral carcinogenesis” (p=1.5х10-4), “PD-L1 expression and PD-1 checkpoint pathway in cancer” (p=5.2х10-4), “NF-kappa B signaling pathway” (p=6.5х10-4) and “Bacterial invasion of epithelial cells” (p=9.6х10-4). Thus, these findings may provide new insights into molecular mechanisms underlying aTRH in patients with chronic kidney disease, e.g., the NF-kappa B signaling pathway may contribute to the development of aTRH through enhanced inflammatory responses.

Extensive Homoplasy but No Evidence of Convergent Evolution of Repeat Numbers at MIRU Loci in Modern Mycobacterium tuberculosis Lineages.

More human deaths have been attributable to Mycobacterium tuberculosis than any other pathogen, and the epidemic is sustained by ongoing transmission. Various typing schemes have been developed to identify strain-specific differences and track transmission dynamics in affected communities, with recent introduction of whole genome sequencing providing the most accurate assessment. Mycobacterial interspersed repetitive unit (MIRU) typing is a family of variable number tandem repeat schemes that have been widely used to study the molecular epidemiology of M. tuberculosis. MIRU typing was used in most well-resourced settings to perform routine molecular epidemiology. Instances of MIRU homoplasy have been observed in comparison with sequence-based phylogenies, limiting its discriminatory value. A fundamental question is whether the observed homoplasy arises purely through stochastic processes, or whether there is evidence of natural selection. We compared repeat numbers at 24 MIRU loci with a whole genome sequence-based phylogeny of 245 isolates representing three modern M. tuberculosis lineages. This analysis demonstrated extensive homoplasy of repeat numbers, but did not detect any evidence of natural selection of repeat numbers, at least since the ancestral branching of the three modern lineages of M. tuberculosis. In addition, we observed good sensitivity but poor specificity and positive predictive values of MIRU-24 to detect clusters of recent transmission, as defined by whole-genome single nucleotide polymorphism analysis. These findings provide mechanistic insight, and support a transition away from VNTR-based typing toward sequence-based typing schemes for both research and public health purposes.

Utilization of Clustered Regularly Interspaced Short Palindromic Repeats to Genotype Escherichia coli Serogroup O80.

The hypervariable nature of clustered regularly interspaced short palindromic repeats (CRISPRs) makes them valuable biomarkers for subtyping and epidemiological investigation of Escherichia coli. Shiga toxin-producing E. coli (STEC) serogroup O80 is one hybrid pathotype that is emerging recently in Europe and is involved in hemolytic uremic syndrome with bacteremia. However, whether STEC O80 strains can be genotyped using CRISPR has not been evaluated. In this study, we aimed to characterize the genetic diversity of 81 E. coli serogroup O80 isolates deposited in the National Center for Biotechnology Information databases using CRISPR typing and to explore the association between virulence potential and CRISPR types (CTs). A total of 21 CTs were identified in 80 O80 strains. CRISRP typing provided discrimination with variants of a single serotype, which suggested a stronger discriminatory power. Based on CRISPR spacer profiles, 70 O80:H2 isolates were further divided into four lineages (lineage LI, LII, LIII, and LIV), which correlated well with whole-genome single nucleotide polymorphisms typing and virulence gene profiles. Moreover, the association between CRISPR lineages and virulence gene profiles hinted that STEC O80:H2 strains may originate from O80:H19 or O80:H26 and that lineage LI may have been evolved from lineage LII. CT2 and CT13 were shared by human and cattle isolates, suggesting that there might be the potential transmission between cattle and human. Collectively, CRISPR typing is one technology that can be used to monitor the transmission of STEC O80 strains and provide new insights into microevolution of serogroup O80.

The evolution of strong reproductive isolation between sympatric intertidal snails.

The evolution of strong reproductive isolation (RI) is fundamental to the origins and maintenance of biological diversity, especially in situations where geographical distributions of taxa broadly overlap. But what is the history behind strong barriers currently acting in sympatry? Using whole-genome sequencing and single nucleotide polymorphism genotyping, we inferred (i) the evolutionary relationships, (ii) the strength of RI, and (iii) the demographic history of divergence between two broadly sympatric taxa of intertidal snail. Despite being cryptic, based on external morphology, Littorina arcana and Littorina saxatilis differ in their mode of female reproduction (egg-laying versus brooding), which may generate a strong post-zygotic barrier. We show that egg-laying and brooding snails are closely related, but genetically distinct. Genotyping of 3092 snails from three locations failed to recover any recent hybrid or backcrossed individuals, confirming that RI is strong. There was, however, evidence for a very low level of asymmetrical introgression, suggesting that isolation remains incomplete. The presence of strong, asymmetrical RI was further supported by demographic analysis of these populations. Although the taxa are currently broadly sympatric, demographic modelling suggests that they initially diverged during a short period of geographical separation involving very low gene flow. Our study suggests that some geographical separation may kick-start the evolution of strong RI, facilitating subsequent coexistence of taxa in sympatry. The strength of RI needed to achieve sympatry and the subsequent effect of sympatry on RI remain open questions. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Breed- and trait-specific associations define the genetic architecture of calving performance traits in cattle.

Reducing the incidence of both the degree of assistance required at calving, as well as the extent of perinatal mortality (PM) has both economic and societal benefits. The existence of heritable genetic variability in both traits signifies the presence of underlying genomic variability. The objective of the present study was to locate regions of the genome, and by extension putative genes and mutations, that are likely to be underpinning the genetic variability in direct calving difficulty (DCD), maternal calving difficulty (MCD), and PM. Imputed whole-genome single-nucleotide polymorphism (SNP) data on up to 8,304 Angus (AA), 17,175 Charolais (CH), 16,794 Limousin (LM), and 18,474 Holstein-Friesian (HF) sires representing 5,866,712 calving events from descendants were used. Several putative quantitative trait loci (QTL) regions associated with calving performance both within and across dairy and beef breeds were identified, although the majority were both breed- and trait-specific. QTL surrounding and encompassing the myostatin (MSTN) gene were associated (P < 5 × 10−8) with DCD and PM in both the CH and LM populations. The well-known Q204X mutation was the fifth strongest association with DCD in the CH population and accounted for 5.09% of the genetic variance in DCD. In contrast, none of the 259 segregating variants in MSTN were associated (P > × 10−6) with DCD in the LM population but a genomic region 617 kb downstream of MSTN was associated (P < 5 × 10−8). The genetic architecture for DCD differed in the HF population relative to the CH and LM, where two QTL encompassing ZNF613 on Bos taurus autosome (BTA)18 and PLAG1 on BTA14 were identified in the former. Pleiotropic SNP associated with all three calving performance traits were also identified in the three beef breeds; 5 SNP were pleiotropic in AA, 116 in LM, and 882 in CH but no SNP was associated with more than one trait within the HF population. The majority of these pleiotropic SNP were on BTA2 surrounding MSTN and were associated with both DCD and PM. Multiple previously reported, but also novel QTL, associated with calving performance were detected in this large study. These also included QTL regions harboring SNP with the same direction of allele substitution effect for both DCD and MCD thus contributing to a more effective simultaneous selection for both traits.

Molecular Characteristics of Brucella Isolates Collected From Humans in Hainan Province, China.

Brucellosis has been reported in several regions of Hainan Province, but the extent of the disease has not been fully elucidated. Conventional biotyping methods, multiple locus variable number tandem repeats analysis (MLVA), and single-nucleotide polymorphisms (SNPs) from draft genome sequencing were employed to characterize the strains. There were four biovars (Brucella melitensis bv. 1, 2, and 3 and Brucella suis bv. 3) detected, which showed that the biovar diversity of Brucella in Hainan is higher than in other areas of China. Both B. melitensis bv. 3 and B. suis bv. 3 were dominant species and showed epidemiology patterns that were compatible with both southern and northern China. Eight of MLVA-11 genotypes were known (31, 111, 116, 120, 136, 291, 297, and 345), and the remaining seven were novel (HN11-1 to HN11-7); these data showed that Brucella strains in this study had multiple geographic origins and exhibited characteristics of origin and evolution of co-existing imported and Hainan specific lineage. A total of 41 strains were found, belonging to 37 unique genotypes that each represented a single strain, which suggests that these strains were not directly related epidemiologically and indicates that the epidemic characteristics of human brucellosis in Hainan was dominated by sporadic strains. The high HGDI values were observed in MLVA-8, MLVA-11, and MLVA-16 among two species, suggesting considerable genetic diversity among these species. MST is characterized based on MLVA-16 that was found both throughout China and on a global level and showed that strains of this study had significant genetic differences with strains from many parts of the globe and seemingly represent a unique genetic lineage. Whole-genome SNP analysis showed that four B. melitensis were closely related to strains from China’s northern provinces, and the source of infection was partly of human brucellosis in this province that may have been from these regions. The B. suis were closely related to strains from the United States, and further investigation of the transportation of animals, such as pigs, is needed to elucidate the origins of these strains.

Identification of Whole-Genome Significant Single Nucleotide Polymorphisms in Candidate Genes Associated With Serum Biochemical Traits in Chinese Holstein Cattle.

A genome-wide association study (GWAS) was conducted on 23 serum biochemical traits in Chinese Holstein cattle. The experimental population consisted of 399 cattle, each genotyped by a commercial bovine 50K SNP chip, which had 49,663 SNPs. After data cleaning, 41,092 SNPs from 361 Holstein cattle were retained for GWAS. The phenotypes were measured values of serum measurements of these animals that were taken at 11 days after parturition. Two statistical models, a fixed-effect linear regression model (FLM) and a mixed-effect linear model (MLM), were used to estimate the association effects of SNPs. Genome-wide significant and suggestive thresholds were set up to be 1.22E−06 and 2.43E−06, respectively. In the Chinese Holstein population, FLM identified 81 genome-wide significant (0.05/41,092 = 1.22E−06) SNPs associated with 11 serum traits. Among these SNPs, five SNPs (BovineHD0100005950, ARS-BFGL-NGS-115158, BovineHD1500021175, BovineHD0800028900, and BTB-00442438) were also identified by the MLM to have genome-wide suggestive effects on CHE, DBIL, and LDL. Both statistical models pinpointed two SNPs that had significant effects on the Holstein population. The SNP BovineHD0800028900 (located near the gene LOC101903458 on chromosome 8) was identified to be significantly associated with serum high- and low-density lipoprotein (HDL and LDL), whereas BovineHD1500021175 (located in 73.4Mb on chromosome 15) was an SNP significantly associated with total bilirubin and direct bilirubin (TBIL and DBIL). Further analyses are needed to identify the causal mutations affecting serum traits and to investigate the correlation of effects for loci associated with fatty liver disease in dairy cattle.

Epidemiological analysis of Arcanobacterium phocae isolated from cases of mink dermatitis of a single farm

The present study was designed to identify nine Arcanobacterium phocae strains isolated from cases of mink dermatitis of a single farm in Finland and characterize the strains for epidemiological relationships. All nine strains and previously described A. phocae used for comparative purposes were identified and further characterized phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), by Fourier Transform Infrared Spectroscopy (FT-IR) and genotypically by detection of phocaelysin encoding gene phl with a previously developed loop-mediated isothermal amplification (LAMP) assay and by sequencing 16S rRNA gene and gene phl, the elongation factor tu encoding gene tuf and the β subunit of bacterial RNA polymerase encoding gene rpoB. Genetic relatedness among isolates was determined using whole-genome single nucleotide polymorphism (wgSNP) analysis. The wgSNP results, partly the MALDI-TOF MS and FT-IR analyses and sequencing of the genes, revealed that the nine A. phocae strains recovered from a single farm showed close sequence similarities among each other and differed from previously investigated A. phocae strains isolated from other farms and animals in Finland and from the A. phocae type strain. This indicated a close epidemiological relationship of the A. phocae strains isolated from a single farm and that the nine A. phocae strains of the present study might have developed from a common ancestor.

Combining Standard Molecular Typing and Whole Genome Sequencing to Investigate Pseudomonas aeruginosa Epidemiology in Intensive Care Units.

Pseudomonas aeruginosa is one of the main pathogens responsible for nosocomial infections, particularly in Intensive Care Units (ICUs). Due to the complexity of P. aeruginosa ecology, only powerful typing methods can efficiently allow its surveillance and the detection during expanding outbreaks. An increase in P. aeruginosa incidence was observed in the ICUs of the Lausanne University Hospital between 2010 and 2014. All clinical and environmental isolates retrieved during this period were typed with Double locus sequence typing (DLST), which detected the presence of three major genotypes: DLST 1–18, DLST 1–21, and DLST 6–7. DLST 1–18 (ST1076) isolates were previously associated with an epidemiologically well-described outbreak in the burn unit. Nevertheless, DLST 1–21 (ST253) and DLST 6–7 (ST17) showed sporadic occurrence with only few cases of possible transmission between patients. Whole genome sequencing (WGS) was used to further investigate the epidemiology of these three major P. aeruginosa genotypes in the ICUs. WGS was able to differentiate between outbreak and non-outbreak isolates and confirm suspected epidemiological links. Additionally, whole-genome single nucleotide polymorphisms (SNPs) results considered isolates as closely related for which no epidemiological links were suspected, expanding the epidemiological investigation to unsuspected links. The combination of a first-line molecular typing tool (DLST) with a more discriminatory method (WGS) proved to be an accurate and cost-efficient typing strategy for the investigation of P. aeruginosa epidemiology in the ICUs.

Inference of Breed Structure in Farm Animals: Empirical Comparison between SNP and Microsatellite Performance.

Knowledge of population structure is essential to improve the management and conservation of farm animal genetic resources. Microsatellites, which have long been popular for this type of analysis, are more and more neglected in favor of whole-genome single nucleotide polymorphism (SNP) chips that are now available for the main farmed animal species. In this study, we compared genetic patterns derived from microsatellites to that inferred by SNPs, considering three pairs of datasets of sheep and cattle. Population genetic differentiation analyses (Fixation index, FST), as well as STRUCTURE analyses showed a very strong consistency between the two types of markers. Microsatellites gave pictures that were largely concordant with SNPs, although less accurate. The best concordance was found in the most complex dataset, which included 17 French sheep breeds (with a Pearson correlation coefficient of 0.95 considering the 136 values of pairwise FST, obtained with both types of markers). The use of microsatellites reduces the cost and the related analyses do not require specific computer equipment (i.e., information technology (IT) infrastructure able to provide adequate computing and storage capacity). Therefore, this tool may still be a very appropriate solution to evaluate, in a first stage, the general state of livestock at national scales. At a time when local breeds are disappearing at an alarming rate, it is urgent to improve our knowledge of them, in particular by promoting tools accessible to the greatest number.

Mutation screening of genes associated with congenital talipes equinovarus in pakistani families

Objectives: Congenital talipes equinovarus (CTEV) is characterized by midfoot cavus, forefoot adducts, hindfoot varus, and equinus. The condition leads to difficulty in walking. CTEV is one of the most common congenital birth defects and an incidence of 1–2/1000 newborns has been reported. The etiology of the CTEV is not completely understood. Multiple etiological factors, both environmental and genetic, have been implicated in the pathogenesis of CTEV. Genetic factors associated with the development of CTEV include mutations in genes related to limb patterning and development of muscle and blood vessels. The present study aims to screen four genes to detect the mutation underlying CTEV in families from Pakistan. Methods: Peripheral blood samples were collected from ten individuals from two Pakistani families with CTEV segregating in an autosomal recessive manner. Genomic DNA was isolated, followed by primer designing and Sanger sequencing of four known genes associated with the isolated form of CTEV (TBX4, PITX1, HOXD12, and HOXD9). Results: Clinical diagnoses of the affected individuals were made by consultant orthopedics. Sequencing analyses revealed that the four candidate genes screened here are not responsible for the CTEV phenotype in both families. Conclusion: Failure to detect sequence variant in four known genes associated with the isolated form of CTEV lead us to the conclusion that TBX4, PITX1, HOXD12, and HOXD9 mutations are not responsible for CTEV in both families. Therefore, genome-wide studies, including whole-genome single nucleotide polymorphism genotyping and whole-exome sequencing, are required to identify the underlying genetic defects in these families.

Multiple methods used for type detection of uniparental disomy in paternity testing.

Uniparental disomy (UPD) has attracted more attention recently in paternity testing, though it is an infrequent genetic event. Although short tandem repeat (STR) profiling has been widely used in paternity testing, it is not sufficient to use STR only to judge the genetic relationship, because the existence of UPD will inevitably affect the results of genotyping. Compared with complete UPD, segmental UPD is more difficult to detect because it does not affect all genotypes on the same chromosome. It is necessary to determine the type of UPD with multiple methods because a single method is not sufficient. Therefore, it is advisable to detect UPD in paternity testing with multiple methods. In this study, after autosomal STR profiling was used, we found that there were several gene loci on the same chromosome that did not conform to Mendelian genetic law, thus we highly suspected the existence of UPD and performed X-STR profiling immediately. Then whole-genome single nucleotide polymorphism (SNP) array analysis was performed to identify the type, and the results provided straightforward evidence for distinguishing complete from segmental UPD. Lastly, we used deletion insertion polymorphism (DIP)-SNP SNaPshot assay and Miseq FGx sequencing (for SNP and STR) to determine whether the mutation source is maternal uniparental disomy (mUPD) or paternal uniparental disomy (pUPD). To avoid false exclusion of kinship, it is vital to determine the type of UPD in paternity testing and effective strategies based on multiple methods to detect the type of UPD are provided in this study.

Antimicrobial resistance genetic factor identification from whole-genome sequence data using deep feature selection

BackgroundAntimicrobial resistance (AMR) is a major threat to global public health because it makes standard treatments ineffective and contributes to the spread of infections. It is important to understand AMR’s biological mechanisms for the development of new drugs and more rapid and accurate clinical diagnostics. The increasing availability of whole-genome SNP (single nucleotide polymorphism) information, obtained from whole-genome sequence data, along with AMR profiles provides an opportunity to use feature selection in machine learning to find AMR-associated mutations. This work describes the use of a supervised feature selection approach using deep neural networks to detect AMR-associated genetic factors from whole-genome SNP data.ResultsThe proposed method, DNP-AAP (deep neural pursuit – average activation potential), was tested on a Neisseria gonorrhoeae dataset with paired whole-genome sequence data and resistance profiles to five commonly used antibiotics including penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime. The results show that DNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae, and also provide a list of candidate genomic features (SNPs) that might lead to the discovery of novel AMR determinants. Logistic regression classifiers were built with the identified SNPs and the prediction AUCs (area under the curve) for penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime were 0.974, 0.969, 0.949, 0.994, and 0.976, respectively.ConclusionsDNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae. It also provides a list of candidate genes and intergenic regions that might lead to novel AMR factor discovery. More generally, DNP-AAP can be applied to AMR analysis of any bacterial species with genomic variants and phenotype data. It can serve as a useful screening tool for microbiologists to generate genetic candidates for further lab experiments.