Whole-genome assembly of a novel invertebrate herpesvirus from the gastropod Babylonia areolata.

Molluscan herpesviruses cause disease in species of major importance to aquaculture and are the only known herpesviruses to infect invertebrates, which lack an adaptive immune system. Understanding the evolution of malacoherpesviruses in relation to their hosts will likely require comparative genomic studies on multiple phylogenetic scales. Currently, only two malacoherpesvirus species have genomes that have been fully assembled, which limits the ability to perform comparative genomic studies on this family of viruses. In the present study, we fully assemble a herpesvirus from Illumina and Nanopore sequence data that were previously used to assemble the genome of the gastropod Babylonia areolata. We tentatively assign this novel herpesvirus to the genus Aurivirus within the family Malacoherpesviridae based on a phylogenetic analysis of DNA polymerase. While structurally similar to other malacoherpesvirus genomes, a synteny analysis of the novel herpesvirus with another Aurivirus species indicates that genomic rearrangements might be an important process in the evolution of this genus. We anticipate that future complete assemblies of malacoherpesviruses will be a valuable resource in comparative herpesvirus research.

Identification of Novel Induced Mutations in Seed and Vegetatively Propagated Plants from Reduced Representation or Whole Genome Sequencing Data.

Treatment of plants with chemical mutagens results primarily in the production of novel single nucleotide variants. Mutagenesis is a mostly random process and as such plants derived from mutagenesis of different seeds or in vitro material are expected to accumulate different mutations. An important step in the creation of a mutant population for forward or reverse genetics is the choice of treatment conditions (e.g., dosage) such that sufficient mutations accumulate while not adversely affecting propagation of the plant. DNA sequencing provides a quick method to evaluate the effect of different treatment conditions and their effect on the density and spectrum of accumulated mutations. Whole genome sequencing or reduced representation sequencing is carried out followed by mapping to a reference genome and production of a Variant Call Format (VCF) file. We provide here a method for generating a multi-sample VCF from mutagenized plants and describe a new tool to streamline the process of recovering unique induced mutations and determining their possible effect on gene function.

[Carbapenemase Genes, Virulence Genes, and Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Derived From Bloodstream Infections]. / è¡€æµæ„ŸæŸ“ç¢³é’éœ‰çƒ¯è€è¯è‚ºç‚Žå ‹é›·ä¼¯èŒçš„è€è¯åŸºå› å’Œæ¯’åŠ›åŸºå› åŠåˆ†å­æµè¡Œç— å­¦ç ”ç©¶.

Objective: To investigate the clinical characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from patients with bloodstream infections in a large tertiary-care general hospital in Southwest China. Methods: A total of 131 strains of non-repeating CRKP were collected from the blood cultures of patients who had bloodstream infections in 2015-2019. The strains were identified by VITEK-2, a fully automated microbial analyzer, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The minimum inhibitory concentration (MIC) was determined by microbroth dilution method. The common carbapenemase resistant genes and virulence factors were identified by PCR. Homology analysis was performed by multilocus sequencing typing. Whole genome sequencing was performed to analyze the genomic characteristics of CRKP without carbapenemase. Results: The 131 strains of CRKP showed resistance to common antibiotics, except for polymyxin B (1.6% resistance rate) and tigacycline (8.0% resistance rate). A total of 105 (80.2%) CRKP strains carried the Klebsiella pneumoniae carbapenemase (KPC) resistance gene, 15 (11.4%) strains carried the New Delhi Metallo-ß-lactamase (NDM) gene, and 4 (3.1%) isolates carried both KPC and NDM genes. Sequence typing (ST) 11 (74.0%) was the dominant sequence type. High detection rates for mrkD (96.2%), fimH (98.5%), entB (100%), and other virulence genes were reported. One hypervirulent CRKP strain was detected. The seven strains of CRKP that did not produce carbapenemase were shown to carry ESBL or AmpC genes and had anomalies in membrane porins OMPK35 and OMPK36, according to whole genome sequencing. Conclusion: In a large-scale tertiary-care general hospital, CRKP mainly carries the KPC gene, has a high drug resistance rate to a variety of antibiotics, and possesses multiple virulence genes. Attention should be paid to CRKP strains with high virulence.

DNA Methylation in Ctenophores.

Epigenomic regulation and dynamic DNA methylation, in particular, are widespread mechanisms orchestrating the genome operation across time and species. Whole-genome bisulfite sequencing (WGBS) is currently the only method for unbiasedly capturing the presence of 5-methylcytosine (5-mC) DNA methylation patterns across an entire genome with single-nucleotide resolution. Bisulfite treatment converts unmethylated cytosines to uracils but leaves methylated cytosines intact, thereby creating a map of all methylated cytosines across a genome also known as a methylome. These epigenomic patterns of DNA methylation have been found to regulate gene expression and influence gene evolution rates between species. While protocols have been optimized for vertebrate methylome production, little adaptation has been done for invertebrates. Creating a methylome reference allows comparisons to be made between rates of transcription and epigenomic patterning in animals. Here we present a method of library construction for bisulfite sequencing optimized for non-bilateral metazoans such as the ctenophore, Mnemiopsis leidyi. We have improved upon our previously published method by including spike-in genomic DNA controls to measure methylation conversion rates. By pooling two bisulfite conversion reactions from the same individual, we also produced sequencing libraries that yielded a higher percentage of sequenced reads uniquely mapping to the reference genome. We successfully detected 5-mC in whole-animal methylomes at CpG, CHG, and CHH sites and visualized datasets using circos diagrams. The proof-of-concept tests were performed both under control conditions and following injury tests with changes in methylation patterns of genes encoding innexins, toxins and neuropeptides. Our approach can be easily adapted to produce epigenomes from other fragile marine animals.

Understanding genetic variability: exploring large-scale copy number variants through non-invasive prenatal testing in European populations.

Large-scale copy number variants (CNVs) are structural alterations in the genome that involve the duplication or deletion of DNA segments, contributing to genetic diversity and playing a crucial role in the evolution and development of various diseases and disorders, as they can lead to the dosage imbalance of one or more genes. Massively parallel sequencing (MPS) has revolutionized the field of genetic analysis and contributed significantly to routine clinical diagnosis and screening. It offers a precise method for detecting CNVs with exceptional accuracy. In this context, a non-invasive prenatal test (NIPT) based on the sequencing of cell-free DNA (cfDNA) from pregnant women's plasma using a low-coverage whole genome MPS (WGS) approach represents a valuable source for population studies. Here, we analyzed genomic data of 12,732 pregnant women from the Slovak (9,230), Czech (1,583), and Hungarian (1,919) populations. We identified 5,062 CNVs ranging from 200 kbp and described their basic characteristics and differences between the subject populations. Our results suggest that re-analysis of sequencing data from routine WGS assays has the potential to obtain large-scale CNV population frequencies, which are not well known and may provide valuable information to support the classification and interpretation of this type of genetic variation. Furthermore, this could contribute to expanding knowledge about the central European genome without investing in additional laboratory work, as NIPTs are a relatively widely used screening method.

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

A Practical Guide to Whole Genome Sequencing in the NICU.

The neonatal period is a peak time for the presentation of genetic disorders that can be diagnosed using whole genome sequencing (WGS). While any one genetic disorder is individually rare, they collectively contribute to significant morbidity, mortality, and health-care costs. As the cost of WGS continues to decline and becomes increasingly available, the ordering of rapid WGS for NICU patients with signs or symptoms of an underlying genetic condition is now feasible. However, many neonatal clinicians are not comfortable with the testing, and unfortunately, there is a dearth of geneticists to facilitate testing for every patient that needs it. Here, we will review the science behind WGS, diagnostic capabilities, limitations of testing, time to consider testing, test initiation, interpretation of results, developing a plan of care that incorporates genomic information, and returning WGS results to families.

Bridging horizons beyond CIRCULATE-Japan: a new paradigm in molecular residual disease detection via whole genome sequencing-based circulating tumor DNA assay.

Circulating tumor DNA (ctDNA) is the fraction of cell-free DNA in patient blood that originates from a tumor. Advances in DNA sequencing technologies and our understanding of the molecular biology of tumors have increased interest in exploiting ctDNA to facilitate detection of molecular residual disease (MRD). Analysis of ctDNA as a promising MRD biomarker of solid malignancies has a central role in precision medicine initiatives exemplified by our CIRCULATE-Japan project involving patients with resectable colorectal cancer. Notably, the project underscores the prognostic significance of the ctDNA status at 4 weeks post-surgery and its correlation to adjuvant therapy efficacy at interim analysis. This substantiates the hypothesis that MRD is a critical prognostic indicator of relapse in patients with colorectal cancer. Despite remarkable advancements, challenges endure, primarily attributable to the exceedingly low ctDNA concentration in peripheral blood, particularly in scenarios involving low tumor shedding and the intrinsic error rates of current sequencing technologies. These complications necessitate more sensitive and sophisticated assays to verify the clinical utility of MRD across all solid tumors. Whole genome sequencing (WGS)-based tumor-informed MRD assays have recently demonstrated the ability to detect ctDNA in the parts-per-million range. This review delineates the current landscape of MRD assays, highlighting WGS-based approaches as the forefront technique in ctDNA analysis. Additionally, it introduces our upcoming endeavor, WGS-based pan-cancer MRD detection via ctDNA, in our forthcoming project, SCRUM-Japan MONSTAR-SCREEN-3.

FinaleMe: Predicting DNA methylation by the fragmentation patterns of plasma cell-free DNA.

Analysis of DNA methylation in cell-free DNA reveals clinically relevant biomarkers but requires specialized protocols such as whole-genome bisulfite sequencing. Meanwhile, millions of cell-free DNA samples are being profiled by whole-genome sequencing. Here, we develop FinaleMe, a non-homogeneous Hidden Markov Model, to predict DNA methylation of cell-free DNA and, therefore, tissues-of-origin, directly from plasma whole-genome sequencing. We validate the performance with 80 pairs of deep and shallow-coverage whole-genome sequencing and whole-genome bisulfite sequencing data.

Implementation of a high-throughput whole genome sequencing approach with the goal of maximizing efficiency and cost effectiveness to improve public health.

This manuscript describes the development of a streamlined, cost-effective laboratory workflow to meet the demands of increased whole genome sequence (WGS) capacity while achieving mandated quality metrics. From 2020 to 2021, the Wadsworth Center Bacteriology Laboratory (WCBL) used a streamlined workflow to sequence 5,743 genomes that contributed sequence data to nine different projects. The combined use of the QIAcube HT, Illumina DNA Prep using quarter volume reactions, and the NextSeq allowed the WCBL to process all samples that required WGS while also achieving a median turn-around time of 7 days (range 4 to 10 days) and meeting minimum sequence quality requirements. Public Health Laboratories should consider implementing these methods to aid in meeting testing requirements within budgetary restrictions. IMPORTANCE: Public Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Population Structure and Selection Signal Analysis of Nanyang Cattle Based on Whole-Genome Sequencing Data.

With a rich breeding history, Nanyang cattle (NY cattle) have undergone extensive natural and artificial selection, resulting in distinctive traits such as high fertility, excellent meat quality, and disease resistance. This makes them an ideal model for studying the mechanisms of environmental adaptability. To assess the population structure and genetic diversity of NY cattle, we performed whole-genome resequencing on 30 individuals. These data were then compared with published whole-genome resequencing data from 432 cattle globally. The results indicate that the genetic structure of NY cattle is significantly different from European commercial breeds and is more similar to North-Central Chinese breeds. Furthermore, among all breeds, NY cattle exhibit the highest genetic diversity and the lowest population inbreeding levels. A genome-wide selection signal analysis of NY cattle and European commercial breeds using Fst, θπ-ratio, and θπ methods revealed significant selection signals in genes associated with reproductive performance and immunity. Our functional annotation analysis suggests that these genes may be responsible for reproduction (MAP2K2, PGR, and GSE1), immune response (NCOA2, HSF1, and PAX5), and olfaction (TAS1R3). We provide a comprehensive overview of sequence variations in the NY cattle genome, revealing insights into the population structure and genetic diversity of NY cattle. Additionally, we identify candidate genes associated with important economic traits, offering valuable references for future conservation and breeding efforts of NY cattle.

Community Voices and Whole-Genome Sequencing for Tuberculosis: Storytelling and the Importance of Listening.

One of the primary public health functions of a tuberculosis (TB) program is to arrest the spread of infection. Traditionally, TB programs have relied on epidemiological information, gathered through contact tracing, to infer that transmission has occurred between people. The ability of drawing such inferences is extensively context dependent. Where epidemiological information has been strong, such as 2 cases of TB occurring sequentially within a single household, confidence in such inferences is high; conversely, public health authorities have been less certain about the significance of TB cases merely occurring in the same wider social group or geographic area. Many current laboratory tests for TB used globally may be sufficient to confirm a diagnosis and guide appropriate therapy but still be insufficiently precise for distinguishing two strains reliably. In short, drawing inferences regarding a chain of transmissions has always been as much art as science.

Characteristic SNPs defining the major multidrug-resistant Mycobacterium tuberculosis clusters identified by EuSeqMyTB to support routine surveillance, EU/EEA, 2017 to 2019.

BackgroundThe EUSeqMyTB project, conducted in 2020, used whole genome sequencing (WGS) for surveillance of drug-resistant Mycobacterium tuberculosis in the European Union/European Economic Area (EU/EEA) and identified 56 internationally clustered multidrug-resistant (MDR) tuberculosis (TB) clones.AimWe aimed to define and establish a rapid and computationally simple screening method to identify probable members of the main cross-border MDR-TB clusters in WGS data to facilitate their identification and track their future spread.MethodsWe screened 34 of the larger cross-border clusters identified in the EuSeqMyTB pilot study (2017-19) for characteristic single nucleotide polymorphism (SNP) signatures that could identify and define members of each cluster. We also linked this analysis with published clusters identified in previous studies and identified more distant genetic relationships between some of the current clusters.ResultsA panel of 30 characteristic SNPs is presented that can be used as an initial (routine) screen for members of each cluster. For four of the clusters, no unique defining SNP could be identified; three of these are closely related (within approximately 20 SNPs) to one or more other clusters and likely represent a single established MDR-TB clade composed of multiple recent subclusters derived from the previously described ECDC0002 cluster.ConclusionThe identified SNP signatures can be integrated into routine pipelines and contribute to the more effective monitoring, rapid and widespread screening for TB. This SNP panel will also support accurate communication between laboratories about previously identified internationally transmitted MDR-TB genotypes.

Structural rearrangements as a recurrent pathogenic mechanism for SETBP1 haploinsufficiency.

Chromosomal structural rearrangements consist of anomalies in genomic architecture that may or may not be associated with genetic material gain and loss. Evaluating the precise breakpoint is crucial from a diagnostic point of view, highlighting possible gene disruption and addressing to appropriate genotype-phenotype association. Structural rearrangements can either occur randomly within the genome or present with a recurrence, mainly due to peculiar genomic features of the surrounding regions. We report about three non-related individuals, harboring chromosomal structural rearrangements interrupting SETBP1, leading to gene haploinsufficiency. Two out of them resulted negative to Chromosomal Microarray Analysis (CMA), being the rearrangement balanced at a microarray resolution. The third one, presenting with a complex three-chromosome rearrangement, had been previously diagnosed with SETBP1 haploinsufficiency due to a partial gene deletion at one of the chromosomal breakpoints. We thoroughly characterized the rearrangements by means of Optical Genome Mapping (OGM) and Whole Genome Sequencing (WGS), providing details about the involved sequences and the underlying mechanisms. We propose structural variants as a recurrent event in SETBP1 haploinsufficiency, which may be overlooked by laboratory routine genomic analyses (CMA and Whole Exome Sequencing) or only partially determined when associated with genomic losses at breakpoints. We finally introduce a possible role of SETBP1 in a Noonan-like phenotype.

Multi-platform whole genome sequencing for tuberculosis clinical and surveillance applications.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis offers valuable insights for tuberculosis (TB) control. High throughput platforms like Illumina and Oxford Nanopore Technology (ONT) are increasingly used globally, although ONT is known for higher error rates and is less established for genomic studies. Here we present a study comparing the sequencing outputs of both Illumina and ONT platforms, analysing DNA from 59 clinical isolates in highly endemic TB regions of Thailand. The resulting sequence data were used to profile the M. tuberculosis pairs for their lineage, drug resistance and presence in transmission chains, and were compared to publicly available WGS data from Thailand (n = 1456). Our results revealed isolates that are predominantly from lineages 1 and 2, with consistent drug resistance profiles, including six multidrug-resistant strains; however, analysis of ONT data showed longer phylogenetic branches, emphasising the technologies higher error rate. An analysis incorporating the larger dataset identified fifteen of our samples within six potential transmission clusters, including a significant clade of 41 multi-drug resistant isolates. ONT's extended sequences also revealed strain-specific structural variants in pe/ppe genes (e.g. ppe50), which are candidate loci for vaccine development. Despite some limitations, our results show that ONT sequencing is a promising approach for TB genomic research, supporting precision medicine and decision-making in areas with less developed infrastructure, which is crucial for tackling the disease's significant regional burden.

Wales Infants' and childreN's Genome Service (WINGS): providing rapid genetic diagnoses for unwell children.

INTRODUCTION: This study reviews the first 3 years of delivery of the first National Health Service (NHS)-commissioned trio rapid whole genome sequencing (rWGS) service for acutely unwell infants and children in Wales. METHODS: Demographic and phenotypic data were prospectively collected as patients and their families were enrolled in the Wales Infants' and childreN's Genome Service (WINGS). These data were reviewed alongside trio rWGS results. RESULTS: From April 2020 to March 2023, 82 families underwent WINGS, with a diagnostic yield of 34.1%. The highest diagnostic yields were noted in skeletal dysplasias, neurological or metabolic phenotypes. Mean time to reporting was 9 days. CONCLUSION: This study demonstrates that trio rWGS is having a positive impact on the care of acutely unwell infants and children in an NHS setting. In particular, the study shows that rWGS can be applied in an NHS setting, achieving a diagnostic yield comparable with the previously published diagnostic yields achieved in research settings, while also helping to improve patient care and management.

Evaluating the efficacy of MEANGS for mitochondrial genome assembly of cartilaginous and ray-finned fish species.

The assembly of complete and circularized mitochondrial genomes (mitogenomes) is essential for population genetics, phylogenetics and evolution studies. Recently, Song et al. developed a seed-free tool called MEANGS for de novo mitochondrial assembly from whole genome sequencing (WGS) data in animals, achieving highly accurate and intact assemblies. However, the suitability of this tool for marine fish remains unexplored. Additionally, we have concerns regarding the overlap sequences in their original results, which may impact downstream analyses. In this Letter to the Editor, the effectiveness of MEANGS in assembling mitogenomes of cartilaginous and ray-finned fish species was assessed. Moreover, we also discussed the appropriate utilization of MEANGS in mitogenome assembly, including the implementation of the data-cut function and circular detection module. Our observations indicated that with the utilization of these modules, MEANGS efficiently assembled complete and circularized mitogenomes, even when handling large WGS datasets. Therefore, we strongly recommend users employ the data-cut function and circular detection module when using MEANGS, as the former significantly reduces runtime and the latter aids in the removal of overlapped sequences for improved circularization. Furthermore, our findings suggested that approximately 2× coverage of clean WGS data was sufficient for MEANGS to assemble mitogenomes in marine fish species. Moreover, due to its seed-free nature, MEANGS can be deemed one of the most efficient software tools for assembling mitogenomes from animal WGS data, particularly in studies with limited species or genetic background information.

A comparative study of structural variant calling in WGS from Alzheimer's disease families.

Detecting structural variants (SVs) in whole-genome sequencing poses significant challenges. We present a protocol for variant calling, merging, genotyping, sensitivity analysis, and laboratory validation for generating a high-quality SV call set in whole-genome sequencing from the Alzheimer's Disease Sequencing Project comprising 578 individuals from 111 families. Employing two complementary pipelines, Scalpel and Parliament, for SV/indel calling, we assessed sensitivity through sample replicates (N = 9) with in silico variant spike-ins. We developed a novel metric, D-score, to evaluate caller specificity for deletions. The accuracy of deletions was evaluated by Sanger sequencing. We generated a high-quality call set of 152,301 deletions of diverse sizes. Sanger sequencing validated 114 of 146 detected deletions (78.1%). Scalpel excelled in accuracy for deletions ≤100 bp, whereas Parliament was optimal for deletions >900 bp. Overall, 83.0% and 72.5% of calls by Scalpel and Parliament were validated, respectively, including all 11 deletions called by both Parliament and Scalpel between 101 and 900 bp. Our flexible protocol successfully generated a high-quality deletion call set and a truth set of Sanger sequencing-validated deletions with precise breakpoints spanning 1-17,000 bp.

Evaluation of a custom designed hybridisation assay for whole genome sequencing of human adenoviruses direct from clinical samples.

BACKGROUND: Human Adenoviruses are a common cause of disease and can cause significant morbidity and mortality in immunocompromised patients. Nosocomial transmission events can occur with whole genome sequencing playing a crucial role. This study evaluates the performance of a custom designed SureSelectXT target enrichment assay based on 14 adenovirus genomes for sequencing direct from clinical samples. METHODS: Modifications were made to the SureSelectXT low input protocol to enhance performance for viral targets. Consensus sequences were generated using an in-house designed three stage bioinformatics pipeline. We assessed, percentage of on target reads, average depth of coverage and percentage genome coverage to determine assay performance across a range of sample matrices. RESULTS: Whole genome sequences were successfully generated for 91.6 % of samples assessed. Adenovirus DNA concentration was a good indicator of enrichment success. Highly specific enrichment was observed with only 6 % of samples showing < 50 % on target reads. Respiratory and faecal samples performed well where bloods showed higher levels of non-specific enrichment likely confounded by low adenovirus DNA concentrations. Protocol performance did not appear impacted by Adenovirus type or species. CONCLUSION: Overall performance of this modified SureSelectXT protocol appears in line with previously published works although there are some confounding factors requiring further investigation. The use of a small RNA bait set has the potential to reduce associated costs which can be prohibitive.

Whole genome sequencing in clinical practice.

Whole genome sequencing (WGS) is becoming the preferred method for molecular genetic diagnosis of rare and unknown diseases and for identification of actionable cancer drivers. Compared to other molecular genetic methods, WGS captures most genomic variation and eliminates the need for sequential genetic testing. Whereas, the laboratory requirements are similar to conventional molecular genetics, the amount of data is large and WGS requires a comprehensive computational and storage infrastructure in order to facilitate data processing within a clinically relevant timeframe. The output of a single WGS analyses is roughly 5 MIO variants and data interpretation involves specialized staff collaborating with the clinical specialists in order to provide standard of care reports. Although the field is continuously refining the standards for variant classification, there are still unresolved issues associated with the clinical application. The review provides an overview of WGS in clinical practice - describing the technology and current applications as well as challenges connected with data processing, interpretation and clinical reporting.