Fetal structural anomalies and birth defects are primarily caused by genetic variants such as chromosomal number abnormalities, copy number variations (CNV), single nucleotide variants (SNV), and small insertions and deletions (indel). Whole-genome sequencing (WGS) based on next-generation sequencing (NGS) as an emerging technology for genetic disease diagnosis can detect the aforementioned types of variants. In recent years, high-depth WGS (> 30×) for prenatal diagnosis has also become available, and proved to be practical for unraveling the genetic etiology of fetal developmental abnormalities. To facilitate clinical practice, test development and preliminary implementation of WGS for diagnosing fetal structural anomalies, we have formulated a consensus over the application of WGS in prenatal diagnosis by compiling previously published consensuses, guidelines, and research findings to provide a guidance on data analysis, reporting recommendations, and consultation of prenatal WGS results.

Objective: To compare the differences between the variation interpretation standards and guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 (The 2015ACMG/AMP guideline) and the Deafness Specialist Group of the Clinical Genome Resource (ClinGen) in 2018 for hereditary hearing loss (Healing loss, HL) issued the expert specification of the variation interpretation guide (The 2018 HL-EP guideline) in evaluating the pathogenicity of OTOF gene variation in patients with auditory neuropathy. Methods: Thirty-eight auditory neuropathy patients with OTOF gene variant were selected as the study subjects (23 males and 15 females, aged 0.3-25.9 years). Using whole-genome sequencing, whole exome sequencing or target region sequencing (Panel) combined with Sanger sequencing, 38 cases were found to carry more than two OTOF mutation sites. A total of 59 candidate variants were independently interpreted based on the 2015 ACMG/AMP guideline and 2018 HL-EP guideline. Compared with the judgment results in 2015 ACMG/AMP guideline, the variants interpreted as lower pathogenic classifications in the 2018 HL-EP guideline were defined as downgraded variants, and the variants regarded as higher pathogenic classifications were defined as upgraded variants. Statistical analysis was conducted using SPSS 20.0. Results: The concordance rate of variant classification between the guidelines was 72.9%(43/59). The 13.6%(8/59) of variants were upgraded and 13.6% (8/59) of variants downgraded in the classifications of the 2018 HL-EP guideline. A couple of rules saw significant differences between the guidelines (PVS1, PM3, PP2, PP3 and PP5). The distribution of pathogenicity of splicing mutation was statistically different (P=0.013). Conclusions: The 2018 HL-EP guideline is inconsistent with the 2015 ACMG/AMP guideline, when judging the pathogenicity of OTOF gene variants in patients with auditory neuropathy. Through the deletion and refinement of evidence and the breaking of solidification thinking, the 2018 HL-EP guideline makes the pathogenicity grading more traceable and improves the credibility.
目的： 对比2015年美国医学遗传学与基因组学学会（American College of Medical Genetics and Genomics，ACMG）和分子病理学会（Association for Molecular Pathology，AMP）发布的变异解读标准与指南（本文中简称2015 ACMG/AMP指南）与2018年临床基因组资源中心（Clinical Genome Resource，ClinGen）耳聋专病小组针对遗传性听力损失（hearing loss，HL）发布的变异解读指南专家规范（本文中简称2018HL专病指南）在评估听神经病患者OTOF基因变异致病性中的异同。 方法： 以38例OTOF基因变异听神经病患者作为研究对象（男23例、女15例，年龄范围0.3~25.9岁），经全基因组重测序、全外显子组测序或目标区域靶向（Panel）测序结合一代Sanger测序验证，38例听神经病患者均检出携带两个以上OTOF变异位点，共计59个候选位点，分别使用2015ACMG/AMP指南以及2018HL专病指南对其致病性进行判断。与2015年指南判断结果相比，2018年指南判断的致病性等级更强定义为升级，更弱定义为降级。采用SPSS 20.0软件进行统计学分析。 结果： 2015 ACMG/AMP指南和2018 HL专病指南的变异分类一致率为72.9%（43/59）。致病性升级变异位点占13.6%（8/59），致病性降级变异位点占13.6%（8/59）。两指南致病性判定不一致主要集中在PVS1、PM3、PP2、PP3以及PP5等级证据的应用上。剪接变异、错义变异、框内插入/缺失以及同义变异致病性分布发生改变，其中剪接变异改变差异具有统计学意义（P=0.013）。 结论： 针对听神经病患者OTOF基因变异进行致病性判断时，2018HL专病指南与2015ACMG/AMP指南存在不一致，2018HL专病指南对证据进行删减及进一步细分，打破常规对于变异类型的固化思维，使得致病性分级更有迹可循，提高可信度。.

Tuberculosis (TB) is a preventable, treatable, and curable disease. However, in 2020, 9∙9 million people were estimated to have developed tuberculosis, and 1.5 million people were estimated to have died from it. Whereas in India, 2.6 million were diagnosed with TB and 436,000 succumbed to TB in 2019. India (26%) is the major contributor to the global drop in TB cases. The COVID-19 pandemic has substantially reduced access to services for the diagnosis and treatment of TB, resulting in an increase in deaths and a reversal in global progress. [1] Presently, TB incidence is falling at a rate of 2% per year, obstructed mainly by the rearing pandemic of drug-resistant tuberculosis (DRTB). Particularly concerning is multi-drug resistant TB (MDRTB), defined as resistance towards isoniazid (INH) and rifampicin (RIF). [2] The World Health Organization (WHO) targeted to reduce worldwide TB incidence by 90% until 2035. (1) Early initiation of effective treatment based on susceptibility patterns of the Mycobacterium tuberculosis complex (MTBC) is considered key to successful TB control in countries with high DRTB incidence. Worldwide MDRTB treatment outcomes are poor, with cure rates less than 60% (2) due to the lack of comprehensive Drug Susceptibility Testing (DST) in most high MDRTB burden countries. This is leading to the inadequate anti-TB activity of the provided regimens (3-5), unlike regimens advised for DST assure optimal results. (6) In addition to resistances to the established regimens, the resistance to the newer DRTB drugs is increasing. On World TB Day 2022, Academy of Advanced Medical Education, Thyrocare Technologies Limited and HyastackAnalytics - IITB along with expert pulmonologist and renowned physicians from India convened for an advisory board meeting in Delhi on 20th March 2022 to discuss the role of Whole Genome Sequencing (WGS) in the diagnosis and management of TB. Objectives and specific topics relating to WGS in MDRTB were discussed, each expert shared their views, which led to a group discussion with a commitment to putting the patient first, and increasing their collective efforts, the organizations recognized that it is possible to make this goal a reality. The organizations involved in the discussion have declared their commitment to engaging in collaborative efforts to tackle DRTB detection efficiently. They advocate for strengthening access to WGS TB services, controlling and preventing TB, improving surveillance and drug resistance management, and investing in research and development. This Round Table serves as a framework to build on and ensure that the goal of ending TB is achievable with WGS services wherever needed. Post discussion, a uniform consensus was said to be arrived if more than 80% board members agreed to the statement. The present paper is the outcome of aspects presented and discussed in the advisory board meeting.

Massively parallel sequencing techniques, such as whole exome sequencing (WES) and whole genome sequencing (WGS), may reveal unsolicited findings (UFs) unrelated to the diagnostic aim. Such techniques are frequently used for diagnostic purposes in pediatric cases of developmental delay (DD). Yet policy guidelines for informed consent and return of UFs are not well equipped to address specific moral challenges that may arise in these children\'s situations.
In previous empirical studies conducted by our research group, we found that it is sometimes uncertain how children with a DD will develop and whether they could come to possess capacities for autonomous decision-making in the future. Parents sometimes felt this brought them into a Catch-22 like situation when confronted with choices about UFs before undergoing WES in trio-analysis (both the parents\' and child\'s DNA are sequenced). An important reason for choosing to consent to WES was to gain more insight into how their child might develop. However, to make responsible choices about receiving or declining knowledge of UFs, some idea of their child\'s future development of autonomous capacities is needed. This undesirable Catch-22 situation was created by the specific policy configuration in which parents were required to make choices about UFs before being sequencing (trio-analysis). We argue that this finding is relevant for reconfiguring current policies for return of UFs for WES/WGS and propose guidelines that encompass two features. First, the informed consent process ought to be staged. Second, differing guidelines are required for withholding/disclosing a UF in cases of DD appropriate to the level of confidence there is about the child\'s future developmental of autonomous capacities.
When combined with a dynamic consent procedure, these two features of our guidelines could help overcome significant moral challenges that present themselves in the situations of children undergoing genomic sequencing for clarifying a DD.

Whole-genome sequencing of viral isolates is critical for informing transmission patterns and for the ongoing evolution of pathogens, especially during a pandemic. However, when genomes have low variability in the early stages of a pandemic, the impact of technical and/or sequencing errors increases. We quantitatively assessed inter-laboratory differences in consensus genome assemblies of 72 matched SARS-CoV-2-positive specimens sequenced at different laboratories in Sydney, Australia. Raw sequence data were assembled using two different bioinformatics pipelines in parallel, and resulting consensus genomes were compared to detect laboratory-specific differences. Matched genome sequences were predominantly concordant, with a median pairwise identity of 99.997%. Identified differences were predominantly driven by ambiguous site content. Ignoring these produced differences in only 2.3% (5/216) of pairwise comparisons, each differing by a single nucleotide. Matched samples were assigned the same Pango lineage in 98.2% (212/216) of pairwise comparisons, and were mostly assigned to the same phylogenetic clade. However, epidemiological inference based only on single nucleotide variant distances may lead to significant differences in the number of defined clusters if variant allele frequency thresholds for consensus genome generation differ between laboratories. These results underscore the need for a unified, best-practices approach to bioinformatics between laboratories working on a common outbreak problem.

暂无摘要。

In our tertiary care center, the reported susceptibility of E. coli blood isolates to amoxicillin/clavulanic acid exceeded 90% in 2005 and showed a progressive decrease to 50% by 2017. In this study, we investigate whether there is a real increase in resistant E. coli strains or if this apparent decline in reported susceptibility might be attributed to the substitution of CLSI by EUCAST guidelines in 2014. We randomly selected 237 E. coli blood isolates (stored at - 80 °C) from 1985 to 2018 and reassessed their MIC values, applying both the CLSI (fixed ratio of clavulanic acid) and EUCAST guidelines (fixed concentration of clavulanic acid). In parallel, the susceptibility of these isolates was retested by disk diffusion, according to the EUCAST guidelines. Whole genome sequencing was successfully performed on 233 of the 237 isolates. In only 130 of the 237 isolates (55.0%), testing according to the EUCAST and CLSI criteria delivered identical MIC values for amoxicillin/clavulanic acid. In 64 of the 237 isolates (27.0%), the MIC values diverged one dilution; in 38 (16.0%), two dilutions; and in five (2.1%), three dilutions. From these 107 discrepant results, testing according to EUCAST methodology revealed more resistant profiles in 93 E. coli strains (94.1%). Also, phenotypical susceptibility testing according to EUCAST guidelines tends to correlate better with the presence of beta-lactamase genes compared to CLSI testing procedure. This study highlights the low agreement between EUCAST and CLSI methodologies when performing MIC testing of amoxicillin/clavulanic acid. More strains are categorized as resistant when EUCAST guidelines are applied. The low agreement between EUCAST and CLSI was confirmed by WGS, since most of EUCAST resistant/CLSI sensitive isolates harbored beta-lactamase genes.

Our current understanding of the natural evolution of RNA viruses comes largely from consensus level genetic analyses which ignore the diverse mutant swarms that comprise within-host viral populations. The breadth and composition of viral mutant swarms impact viral fitness and adaptation, and the capacity for swarm plasticity is likely to be particularly important for arthropod-borne viruses (arboviruses) that cycle between taxonomically divergent hosts. Despite this, characterization of the relationship between the selective pressures and genetic signatures of the mutant swarm and consensus sequences is lacking. To clarify this, we analyzed previously generated whole genome, deep-sequencing data from 548 West Nile virus samples isolated from avian tissues or mosquitoes in New York State from 1999-2018. Both consensus level (interhost) and minority level (intrahost) nucleotide and amino acid sequences were analyzed, and diversity at each position was calculated across the genome in order to assess the relationship between minority and consensus sequences for individual genes and hosts. Our results indicate that consensus sequences are an inept representation of the overall genetic diversity. Unique host and gene-specific signatures and selective pressures were identified. These data demonstrate that an accurate and comprehensive understanding of arbovirus evolution and adaptation within and between hosts requires consideration of minority genotypes.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a threat to the human population and has created a worldwide pandemic. Daily thousands of people are getting affected by the SARS-CoV-2 virus; India being no exception. In this situation, there is no doubt that vaccine is the primary prevention strategy to contain the wave of COVID-19 pandemic. In this regard, genome-wide analysis of SARS-CoV-2 is important to understand its genetic variability. This has motivated us to analyse 566 Indian SARS-CoV-2 sequences using multiple sequence alignment techniques viz. ClustalW, MUSCLE, ClustalO and MAFFT to align and subsequently identify the lists of mutations as substitution, deletion, insertion and SNP. Thereafter, a consensus of these results, called as Consensus Multiple Sequence Alignment (CMSA), is prepared to have the final list of mutations so that the advantages of all four alignment techniques can be preserved. The analysis shows 767, 2025 and 54 unique substitutions, deletions and SNPs in Indian SARS-CoV-2 genomes. More precisely, out of 54 SNPs, 4 SNPs are present close to the 60% of the virus population. The results of this experiment can be useful for virus classification, designing and defining the dose of vaccine for the Indian population.

Strongyloidiasis is a soil-borne helminthiasis, which, in spite of the up to 370 million people currently estimated to be infected with its causing agent, the nematode Strongyloides stercoralis, is frequently overlooked. Recent molecular taxonomic studies conducted in Southeast Asia and Australia, showed that dogs can carry the same genotypes of S. stercoralis that also infect humans, in addition to a presumably dog-specific Strongyloides species. This suggests a potential for zoonotic transmission of S. stercoralis from dogs to humans. Although natural S. stercoralis infections have not been reported in any host other than humans, non-human primates and dogs, other as yet unidentified animal reservoirs cannot be excluded. Molecular studies also showed that humans carry rather different genotypes of S. stercoralis. As a result, their taxonomic status and the question of whether they differ in their pathogenic potential remains open. It would therefore be very important to obtain molecular genetic/genomic information about S. stercoralis populations from around the world. One way of achieving this (with little additional sampling effort) would be that people encountering S. stercoralis in the process of their diagnostic work preserve some specimens for molecular analysis. Here we provide a guideline for the isolation, preservation, genotyping at the nuclear 18S rDNA and the mitochondrial cox1 loci, and for whole genome sequencing of single S. stercoralis worms. Since in many cases the full analysis is not possible or desired at the place and time where S. stercoralis are found, we emphasize when and how samples can be preserved, stored and shipped for later analysis. We hope this will benefit and encourage researchers conducting field studies or diagnostics to collect and preserve S. stercoralis for molecular genetic/genomic analyses and either analyze them themselves or make them available to others for further analysis.