Application Of Whole Genome Sequencing Research Articles

Application of whole genome sequencing in tuberculosis clinical trials

Application of whole genome sequencing in tuberculosis clinical trials

Global mapping of cancers: The Cancer Genome Atlas and beyond.

Cancer genomes have been explored from the early 2000s through massive exome sequencing efforts, leading to the publication of The Cancer Genome Atlas in 2013. Sequencing techniques have been developed alongside this project and have allowed scientists to bypass the limitation of costs for whole‐genome sequencing (WGS) of single specimens by developing more accurate and extensive cancer sequencing projects, such as deep sequencing of whole genomes and transcriptomic analysis. The Pan‐Cancer Analysis of Whole Genomes recently published WGS data from more than 2600 human cancers together with almost 1200 related transcriptomes. The application of WGS on a large database allowed, for the first time in history, a global analysis of features such as molecular signatures, large structural variations and noncoding regions of the genome, as well as the evaluation of RNA alterations in the absence of underlying DNA mutations. The vast amount of data generated still needs to be thoroughly deciphered, and the advent of machine‐learning approaches will be the next step towards the generation of personalized approaches for cancer medicine. The present manuscript wants to give a broad perspective on some of the biological evidence derived from the largest sequencing attempts on human cancers so far, discussing advantages and limitations of this approach and its power in the era of machine learning.

Leveraging three-dimensional chromatin architecture for effective reconstruction of enhancer-target gene regulatory interactions.

A growing amount of evidence in literature suggests that germline sequence variants and somatic mutations in non-coding distal regulatory elements may be crucial for defining disease risk and prognostic stratification of patients, in genetic disorders as well as in cancer. Their functional interpretation is challenging because genome-wide enhancer–target gene (ETG) pairing is an open problem in genomics. The solutions proposed so far do not account for the hierarchy of structural domains which define chromatin three-dimensional (3D) architecture. Here we introduce a change of perspective based on the definition of multi-scale structural chromatin domains, integrated in a statistical framework to define ETG pairs. In this work (i) we develop a computational and statistical framework to reconstruct a comprehensive map of ETG pairs leveraging functional genomics data; (ii) we demonstrate that the incorporation of chromatin 3D architecture information improves ETG pairing accuracy and (iii) we use multiple experimental datasets to extensively benchmark our method against previous solutions for the genome-wide reconstruction of ETG pairs. This solution will facilitate the annotation and interpretation of sequence variants in distal non-coding regulatory elements. We expect this to be especially helpful in clinically oriented applications of whole genome sequencing in cancer and undiagnosed genetic diseases research.

Mutational signatures in T-lymphocytes of rats treated with N-propyl-N-nitrosourea and procarbazine.

We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T-cells of rats treated in vivo with N-propyl-N-nitrosourea (PNU) or procarbazine (PCZ). The signatures from the treated rats were different from the signature of background mutations. The main component of the spontaneous T-cell mutational signature was C➔T transition with all other single base substitutions evenly distributed. The PNU-induced mutational signature showed relatively equal contributions from C➔T and T➔C transitions, and T➔A transversions. The PCZ-induced signature was characterized by T➔C transitions, T➔A and, to a smaller extent, T➔G transversions. C➔G transversions were infrequent in either the PNU or PCZ signatures. WGS not only allowed mutational signature detection, but also measured quantitative responses to mutagen treatment: 10-40× increases in the number of mutations per clone were detected in T-cell clones from treated rats. The overall strand specificity of induced mutations for annotated rat genes was comparable to the strand specificity of mutations determined previously for the endogenous X-linked Pig-a gene. Our results provide valuable reference data for future applications of WGS in safety research and risk assessment.

Whole-Genome Sequencing Investigation of a Large Nosocomial Outbreak Caused by ST131 H30Rx KPC-Producing Escherichia coli in Italy.

KPC-producing Escherichia coli (KPC-Ec) remains uncommon, being mainly reported as the cause of sporadic episodes of infection rather than outbreak events. Here we retrospectively describe the dynamics of a large hospital outbreak sustained by KPC-Ec, involving 106 patients and 25 hospital wards, during a six-month period. Twenty-nine representative KPC-Ec isolates (8/29 from rectal swabs; 21/29 from other clinical specimens) have been investigated by Whole-Genome Sequencing (WGS). Outbreak isolates showed a multidrug-resistant profile and harbored several resistance determinants, including blaCTX-M-27, aadA5, dfrA17, sulI, gyrA1AB and parC1aAB. Phylogenomic analysis identified the ST131 cluster 1 (23/29 isolates), H30Rx clade C, as responsible for the epidemic event. A further two KPC-Ec ST131 clusters were identified: cluster 2 (n = 2/29) and cluster 3 (n = 1/29). The remaining KPC-Ec resulted in ST978 (n = 2/29) and ST1193 (n = 1/29), and were blaKPC-3 associated. The KPC-Ec ST131 cluster 1, originated in a previous KPC-Kp endemic context probably by plasmid transfer, and showed a clonal dissemination strategy. Transmission of the blaKPC gene to the globally disseminated high-risk ST131 clone represents a serious cause of concern. Application of WGS in outbreak investigations could be useful to better understand the evolution of epidemic events in order to address infection control and contrast interventions, especially when high-risk epidemic clones are involved.

Aspergillus fumigatus and aspergillosis: From basics to clinics.

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.

EP469 - Design of a web-based, participatory education program on clinical applications of whole genome sequencing

eP469 - Design of a web-based, participatory education program on clinical applications of whole genome sequencing

Prevalence and genomic investigation of Salmonella isolates recovered from animal food-chain in Xinjiang, China

Salmonella is a major foodborne pathogen worldwide, causing serious cases of morbidity and mortality due to the consumption of contaminated foods. Animal-borne foods were considered the main source of transferring Salmonella to humans; however, route surveillance by genomic platforms along the food-chain is limited in China. Here, we proceeded to the application of whole genome sequencing in the epidemiological analysis of Salmonella isolated along the food-chain in Xinjiang, China. A total of 2408 samples were collected from farms, slaughterhouses, and markets, and subjected to the isolation of Salmonella strains. 314 (13.04%) of the samples were positive for Salmonella. Phenotypic antimicrobial resistance was conducted by the broth dilution method using 14 antimicrobial agents belonging to ten classes for all 314 isolates. A selection of representative 103 isolates was subjected to whole-genome sequencing for understanding the Salmonella diversity, including serovars, antimicrobial and virulence genes, plasmid types, multi-locus sequence types, and allelic types. We found that S. Agona was the dominant serovar and O:4(B) was the dominant serogroup. The dominant genotype was ST13 and each serovar has a unique MLST pattern. Plasmids prediction reported Col(MGD2)_1 and Col(Ye4449)_1 as the dominant plasmids, in addition to the detection of IncFII(S)_1 and IncFIB(S)_1 carried by all S. Enteritidis isolates. Importantly, virulence genes prediction showed the presence of cdtB gene encoding typhoid toxins, spv genes, and pef gene cluster encoding fimbriae in the genomes of S. Indiana and S. Enteritidis. Phenotypic antimicrobial resistance identified 92.04% of the sampled isolates as multi-drug resistance (MDR), with high resistance to tetracycline (78.03%; 245/314), amoxicillin/ clavulanic acid (75.80%; 238/314), and ampicillin (70.70%; 222/314). Together, we firstly reported the prevalence of MDR Salmonella isolates harboring critical virulence factors transmission via animal-borne food-chain in Xinjiang, hence route surveillance by whole-genome sequencing platform could facilitate recognition and project early warning for the emerging MDR clones along the food-chain.

Whole-genome sequence analysis and comparisons between drug-resistance mutations and minimum inhibitory concentrations of Mycobacterium tuberculosis isolates causing M/XDR-TB

Drug resistance (DR) remains a major challenge for tuberculosis (TB) control. Whole-genome sequencing (WGS) provides the highest genetic resolution for genotypic drug-susceptibility tests (DST). We compared DST profiles of 60 Mycobacterium tuberculosis isolates which were drug resistant according to agar proportion tests (one poly DR-TB, 34 multidrug-resistant TB and 25 extensively drug-resistant TB). We additionally performed minimum inhibitory concentration (MIC) tests using Sensititre MYCOTBI plates (MYCOTB) and a WGS-based DST. Agreement between WGS-based DST and MYCOTB was high for all drugs except ethambutol (65%) and ethionamide (62%). Isolates harboring the -15 c/t inhA promoter mutation had a significantly lower MIC for isoniazid than did isolates with the katG Ser315Thr mutation (p < 0.001). Similar patterns were seen for ethambutol (embB Gly406Asp vs. embB Met306Ile), streptomycin (gid Gly73Ala vs. rpsL Lys43Arg), moxifloxacin (gyrA Ala90Val vs. gyrA Asp94Gly) and rifabutin (rpoB Asp435Phe/Tyr/Val vs. rpoB Ser450Leu). For genotypic heteroresistance, isolates with lower proportion of mapped read tended to has lower MIC of anti-TB drugs than those with higher proportion. These results emphasize the high applicability of WGS for determination of DR-TB and the association of particular mutations with MIC levels.

Virulence and resistance genes profiles and clonal relationships of non-typhoidal food-borne Salmonella strains isolated in Tunisia by whole genome sequencing

Whole genome sequencing (WGS) has made impressive progress in the field of molecular biology. Its most common application for public health is in the area of surveillance of food-borne diseases. WGS has the potential for providing a large amount of information, such as the identification of the strain type, the characterization of antibiotic resistance and virulence, and phylogeny. In our study, thirty-nine non-typhoidal Salmonella strains were isolated from diverse sources in Tunisia. Non-typhoidal Salmonella are among the most common pathogens contaminating food animals. The presence of virulence and antimicrobial resistance determinants in those strains were investigated using whole genome sequencing (WGS) and appropriate data analysis. The genomes were screened for several Salmonella virulence genes using the Virulence Factor Database VFDB. Twelve different virulence profiles, which correspond to the 12 identified serovars, were recognized. Several antimicrobial resistance genes were also detected: aac (6′)-Iaa, sul1, tetA, bla-TEM and qnrS genes. Phylogenetic relationships among the strains were further assessed by a cgMLST analysis. The resulting phylogenetic tree consisted of several clusters consistently with the in silico multilocus sequence typing (MLST) and serotyping. Our findings demonstrated that WGS and subsequent data analysis provided an accurate tool for genetic characterization of bacterial strains compared to usual molecular typing techniques. To the best of our knowledge, this is the first report of an application of WGS for genetic characterization of food-borne Tunisian strains.

A Review of Gene Sequencing in Infertility

Assisted reproductive technology (ART) is the technology used primarily for infertility treatments in order to achieve pregnancy. With the increasing demands of preimplantation genetic testing (PGT), the safety inspection of in-vitro fertilization (IVF), the prevention of pathogenesis in sterility, the application of gene sequencing technology in ART, and the diagnosis of infertility-related diseases have significantly increased. The development and principles of gene sequencing technology, the application of gene sequencing in PGT, the application of whole genome sequencing in ART-conceived children, and the application of whole genome sequencing in infertility-related disorders have been summarized in this review.

The Characteristic of Virulence, Biofilm and Antibiotic Resistance of Klebsiella pneumoniae

Klebsiella pneumoniae is an important gram-negative opportunistic pathogen that causes a variety of infectious diseases, including urinary tract infections, bacteremia, pneumonia, and liver abscesses. With the emergence of multidrug-resistant (MDR) and hypervirulent K. pneumoniae (hvKP) strains, the rapid spread of these clinical strains in geography is particularly worrying. However, the detailed mechanisms of virulence and antibiotic resistance in K. pneumoniae are still not very clear. Therefore, studying and elucidating the pathogenic mechanisms and drug resistance mechanism of K. pneumoniae infection are important parts of current medical research. In this paper, we systematically summarized the virulence, biofilm, and antibiotic tolerance mechanisms of K. pneumoniae, and explored the application of whole genome sequencing and global proteomics, which will provide new clues for clinical treatment of K. pneumoniae.

Application of Whole Genome Sequencing and Pan-Family Multi-Locus Sequence Analysis to Characterize Relationships Within the Family Brucellaceae.

The bacterial family Brucellaceae is currently composed of seven genera, including species of the genus Brucella, a number of which are significant veterinary and zoonotic pathogens. The bacteriological identification of pathogenic Brucella spp. may be hindered by their close phenotypic similarity to other members of the Brucellaceae, particularly of the genus Ochrobactrum. Additionally, a number of novel atypical Brucella taxa have recently been identified, which exhibit greater genetic diversity than observed within the previously described species, and which share genomic features with organisms outside of the genus. Furthermore, previous work has indicated that the genus Ochrobactrum is polyphyletic, raising further questions regarding the relationship between the genus Brucella and wider Brucellaceae. We have applied whole genome sequencing (WGS) and pan-family multi-locus sequence analysis (MLSA) approaches to a comprehensive panel of Brucellaceae type strains, in order to characterize relationships within the family. Phylogenies based on WGS core genome alignments were able to resolve phylogenetic relationships of 31 non-Brucella spp. type strains from within the family, alongside type strains of twelve Brucella species. A phylogeny based on concatenated pan-family MLSA data was largely consistent with WGS based analyses. Notably, recently described atypical Brucella isolates were consistently placed in a single clade with existing species, clearly distinct from all members of the genus Ochrobactrum and wider family. Both WGS and MLSA methods closely grouped Brucella spp. with a sub-set of Ochrobactrum species. However, results also confirmed that the genus Ochrobactrum is polyphyletic, with seven species forming a separate grouping. The pan-family MLSA scheme was subsequently applied to a panel of 50 field strains of the family Brucellaceae, isolated from a wide variety of sources. This analysis confirmed the utility of the pan-Brucellaceae MLSA scheme in placing field isolates in relation to recognized type strains. However, a significant number of these isolates did not cluster with currently identified type strains, suggesting the existence of additional taxonomic diversity within some members of the Brucellaceae. The WGS and pan-family MLSA approaches applied here provide valuable tools for resolving the identity and phylogenetic relationships of isolates from an expanding bacterial family containing a number of important pathogens.

Whole-genome sequencing analysis of multidrug-resistant Mycobacterium tuberculosis from Java, Indonesia.

Introduction. Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem globally, including in Indonesia. Whole-genome sequencing (WGS) analysis has rarely been used for the study of TB and MDR-TB in Indonesia.Aim. We evaluated the use of WGS for drug-susceptibility testing (DST) and to investigate the population structure of drug-resistant Mycobacterium tuberculosis in Java, Indonesia.Methodology. Thirty suspected MDR-TB isolates were subjected to MGIT 960 system (MGIT)-based DST and to WGS. Phylogenetic analysis was done using the WGS data. Results obtained using MGIT-based DST and WGS-based DST were compared.Results. Agreement between WGS and MGIT was 93.33 % for rifampicin, 83.33 % for isoniazid and 76.67 % for streptomycin but only 63.33 % for ethambutol. Moderate WGS-MGIT agreement was found for second-line drugs including amikacin, kanamycin and fluoroquinolone (73.33-76.67 %). MDR-TB was more common in isolates of the East Asian Lineage (63.3%). No evidence of clonal transmission of DR-TB was found among members of the tested population.Conclusion. Our study demonstrated the applicability of WGS for DST and molecular epidemiology of DR-TB in Java, Indonesia. We found no transmission of DR-TB in Indonesia.

Microevolution of Campylobacter jejuni during long-term infection in an immunocompromised host

Campylobacteriosis typically manifests as a short-lived, self-limiting gastrointestinal infection in humans, however prolonged infection can be seen in cases with underlying immunodeficiency. Public Health England received 25 isolates of Campylobacter jejuni from an individual with combined variable immunodeficiency over a period of 15 years. All isolates were typed and archived at the time of receipt. Whole genome sequencing (WGS) and antimicrobial susceptibility testing were performed to examine the relatedness of the isolates and to investigate the changes in the genome that had taken place over the course of the infection. Genomic typing methods were compared to conventional phenotypic methods, and revealed that the infection was caused by a single, persistent strain of C. jejuni belonging to clonal complex ST-45, with evidence of adaptation and selection in the genome over the course of the infection. Genomic analysis of sequence variants associated with antimicrobial resistance identified the genetic background behind rRNA gene mutations causing variable levels of resistance to erythromycin. This application of WGS to examine a persistent case of campylobacteriosis provides insight into the mutations and selective pressures occurring over the course of an infection, some of which have important clinical relevance.

Quantitative microbial risk assessment for Salmonella: Inclusion of whole genome sequencing and genomic epidemiological studies, and advances in the bioinformatics pipeline

Despite the implementation of extensive measures to control food-borne illness caused by Salmonella since the beginning of the 20th century, it remains one of the most important and widespread food-borne pathogens. Quantitative microbial risk assessment (QMRA) has become essential to evaluating and mitigating the public health risk of Salmonella. However, current QMRA models do not incorporate the minute changes in microbial behavior arising from differences in the microbial genome. Genomic analysis, particularly whole genome sequencing (WGS), has undergone significant progress over the past two decades, facilitating rapid data accumulation. This presents an unprecedented opportunity to incorporate genomic data into a QMRA for Salmonella. This review aims to explore the applicability of WGS and other genomic technologies in Salmonella QMRA, and to outline the added value and current limitations and promise of genomics, particularly bacterial WGS, in supporting the integration of this data in a framework to assess the risk of Salmonella. Our review indicated that although comparative genomic studies for Salmonella using WGS has seen a considerable amount of interest over the past decade, the incorporation of genomic data into QMRA remains a difficult and arduous prospect. However, with the progress in bioinformatics and genomic epidemiology, genomic data could certainly be utilized in QMRA to improve and update risk assessment models and risk management strategies for Salmonella, as well as other foodborne pathogens of interest.

Mycobacterium bovis: From Genotyping to Genome Sequencing.

Mycobacterium bovis is the main pathogen of bovine, zoonotic, and wildlife tuberculosis. Despite the existence of programs for bovine tuberculosis (bTB) control in many regions, the disease remains a challenge for the veterinary and public health sectors, especially in developing countries and in high-income nations with wildlife reservoirs. Current bTB control programs are mostly based on test-and-slaughter, movement restrictions, and post-mortem inspection measures. In certain settings, contact tracing and surveillance has benefited from M. bovis genotyping techniques. More recently, whole-genome sequencing (WGS) has become the preferential technique to inform outbreak response through contact tracing and source identification for many infectious diseases. As the cost per genome decreases, the application of WGS to bTB control programs is inevitable moving forward. However, there are technical challenges in data analyses and interpretation that hinder the implementation of M. bovis WGS as a molecular epidemiology tool. Therefore, the aim of this review is to describe M. bovis genotyping techniques and discuss current standards and challenges of the use of M. bovis WGS for transmission investigation, surveillance, and global lineages distribution. We compiled a series of associated research gaps to be explored with the ultimate goal of implementing M. bovis WGS in a standardized manner in bTB control programs.

Application of whole-genome sequencing in a case study of renal tuberculosis in a child

BackgroundUrogenital tuberculosis (TB) is rare in children and usually develops due to reactivation of the foci in the genitourinary tract after the latency period following initial infection. Urogenital TB in children has no pathognomonic clinical features that can result in overlooking or misdiagnosing this clinical entity. Here, we report important findings regarding the pathogenesis and transmission of TB by using genotyping and whole-genome sequencing (WGS) in a study of renal TB case in a child.Case presentationA 13-year-old boy was admitted to the hospital because of high fever, severe dry cough, flank pain and painful urination. Abdominal ultrasonography and CT revealed an 8 mm calculus in the kidney, and clinical findings were initially interpreted as nephrolithiasis. Nevertheless, due to the atypical clinical presentation of kidney stone disease, additional investigations for possible TB were performed. The QuantiFERON®-TB Gold Plus test was positive, and the Mantoux test resulted in 15 mm of induration, confirming infection with Mycobacterium tuberculosis (Mtb). Chest X-ray was normal. Chest CT revealed calcified intrathoracic lymph nodes. The urine sample tested positive for acid-fast bacilli, and Mtb cultures were obtained from urine and bronchial aspirate samples, resulting in a final diagnosis of intrathoracic lymph node and renal TB. Contact investigation revealed that the child’s father was diagnosed with TB when the child was 1 year old. Genotyping and WGS analysis of Mtb isolates of the child and his father confirmed the epidemiological link and pointed to the latency of infection in the child.ConclusionsThis case report confirmed the development of active TB from calcified lesions in adolescent after 12 years of exposure, demonstrated the absence of microevolutionary changes in the Mtb genome during the period of latency, and proved the importance of appropriate evaluation and management to prevent the progression of TB infection to active TB disease. The use of WGS provided the ultimate resolution for the detection of TB transmission and reactivation events.

Resistance Sniffer: An online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using next generation sequencing data

The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the problems that are associated with current phenotypic drug susceptibility testing. However, the application of WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up.ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial genome datasets in different file formats including raw Illumina fastq read files. The program was validated on sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.

Whole genome sequencing reveals genetic diversity in Mycobacterium avium subspecies paratuberculosis population circulating in Irish cattle

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s Disease (JD), a chronic enteritis, in cattle. Whole genome sequencing (WGS) has been applied to many pathogen systems, where its unprecedented resolution has greatly enhanced our understanding of the molecular epidemiology of pathogen transmission. However, WGS has seen limited application to MAP; understanding the transmission dynamics of MAP will inform control of JD. We report the first study into the application of WGS to MAP in Ireland. DNA was extracted from 167 MAP isolates sourced from cattle across Ireland. Libraries were prepared and sequenced on an Illumina-NextSeq500 platform. Sequencing data were processed using an in-house bioinformatic pipeline, which trimmed reads, aligned them to the reference genome MAP K10, followed by variant calling, quality filtering and construction of a maximum-likelihood phylogeny. DNA extracts were also used for MIRU-VNTR typing. The resulting phylogeny shows that the MAP population present in Irish cattle is genetically diverse, which may have resulted from importation of MAP strains from across Europe into Ireland. Similar diversity was observed in a WGS study that noted the impact of cattle imports on the Canadian MAP population. Some Irish isolates were genetically similar to European and Canadian isolates. Comparing our WGS data with MIRU-VNTR indicates that MIRU-VNTR has limited resolution for discriminating MAP strains, and often does not distinguish isolates to sufficient resolution. The genomic data presented here provide the first snapshot of genetic diversity of Irish MAP and a baseline for future studies into spread and persistence of MAP in Irish cattle.