Whole exome sequencing (WES) is a cost-effective method that identifies clinical variants but it demands accurate variant caller tools. Currently available tools have variable accuracy in predicting specific clinical variants. But it may be possible to find the best combination of aligner-variant caller tools for detecting accurate single nucleotide variants (SNVs) and small insertion and deletion (InDels) separately. Moreover, many important aspects of InDel detection are overlooked while comparing the performance of tools, particularly its base pair length. We assessed the performance of variant calling pipelines using the combinations of four variant callers and five aligners on human NA12878 and simulated exome data. We used high confidence variant calls from Genome in a Bottle (GiaB) consortium for validation, and GRCh37 and GRCh38 as the human reference genome. Based on the performance metrics, both BWA and Novoalign aligners performed better with DeepVariant and SAMtools callers for detecting SNVs, and with DeepVariant and GATK for InDels. Furthermore, we obtained similar results on human NA24385 and NA24631 exome data from GiaB. In this study, DeepVariant with BWA and Novoalign performed best for detecting accurate SNVs and InDels. The accuracy of variant calling was improved by merging the top performing pipelines. The results of our study provide useful recommendations for analysis of WES data in clinical genomics.

Summary: By combining X‐ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor‐dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG‐like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram‐positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell–cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram‐positive fimbriae.

The spectrum of RB1gene mutations in Retinoblastoma (RB) patients and the necessity of multiple traditional methods for complete variant analysis make the molecular diagnosis a cumbersome, labor-intensive and time-consuming process. Here, we have used targeted next generation sequencing (NGS) approach with in-house analysis pipeline to explore its potential for the molecular diagnosis of RB. Thirty-three patients with RB and their family members were selected randomly. DNA from patient blood and/or tumor was used for RB1 gene targeted sequencing. The raw reads were obtained from Illumina Miseq. An in-house bioinformatics pipeline was developed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels) and to distinguish between somatic and germline mutations. In addition, ExomeCNV and Cn. MOPS were used to detect copy number variations (CNVs). The pathogenic variants were identified with stringent criteria, and were further confirmed by conventional methods and cosegregation in families. Using our approach, an array of pathogenic variants including SNVs, InDels and CNVs were detected in 85% of patients. Among the variants detected, 63% were germline and 37% were somatic. Interestingly, nine novel pathogenic variants (33%) were also detected in our study. We demonstrated for the first time that targeted NGS is an efficient approach for the identification of wide spectrum of pathogenic variants in RB patients. This study is helpful for the molecular diagnosis of RB in a comprehensive and time-efficient manner.

Purpose: MicroRNAs (miRNAs) are small, stable, noncoding RNA molecules with regulatory function and marked tissue specificity that posttranscriptionally regulate gene expression. However, their role in fungal keratitis remains unknown. The purpose of this study was to identify the miRNA profile and its regulatory role in fungal keratitis. Methods: Normal donor (n = 3) and fungal keratitis (n = 5) corneas were pooled separately, and small RNA deep sequencing was performed using a sequencing platform. A bioinformatics approach was applied to identify differentially-expressed miRNAs and their targets, and select miRNAs were validated by real-time quantitative PCR (qPCR). The regulatory functions of miRNAs were predicted by combining miRNA target genes and pathway analysis. The mRNA expression levels of select target genes were further analyzed by qPCR. Results: By deep sequencing, 75 miRNAs were identified as differentially expressed with fold change greater than 2 and probability score greater than 0.9 in fungal keratitis corneas. The highly dysregulated miRNAs (miR-511-5p, miR-142-3p, miR-155-5p, and miR-451a) may regulate wound healing as they were predicted to specifically target wound inflammatory genes. Moreover, the increased expression of miR-451a in keratitis correlated with reduced expression of its target, macrophage migration inhibitory factor, suggesting possible regulatory functions. Conclusions: This is, to our knowledge, the first report on comprehensive human corneal miRNA expression profile in fungal keratitis. Several miRNAs with high expression in fungal keratitis point toward their potential role in regulation of pathogenesis. Further insights in understanding their role in corneal wound inflammation may help design new therapeutic strategies.

Global emergence of multidrug resistant (MDR) strains limits therapeutic efficacy in Pseudomonas aeruginosa corneal ulcers. Identifying the primary causal factors of resistance shall improve clinical management. In this study, we sought to identify the underlying mechanisms of fluoroquinolone and aminoglycoside resistance in MDR, non-MDR, and drug susceptible P. aeruginosa ( n = 19) strains obtained from keratitis patients. Phenotypic assays were performed to study the bacterial growth kinetics, efflux, permeability, and biofilm formation. Mutational alteration of target genes (DNA sequencing), relative expression of efflux system genes (real time PCR), and detection of aminoglycoside modifying enzyme (AME) genes (PCR) were done by molecular methods. We repeatedly found the mutations in quinolone resistance determining region of fluoroquinolone target genes, gyrA and parC , and the presence of AME genes, aph(3″)-I and aph(6)-I , in all MDR isolates. Furthermore, the MDR isolates were largely characterized by slower growth, cytotoxic type III secretion system genotype, better biofilm-forming ability, and the presence of additional AME genes. The non-MDR isolates were resensitized upon inhibition of active efflux or enhancement of membrane permeability. Altogether this study highlights target gene alteration and enzymatic drug modification as the major mechanisms of quinolone and aminoglycoside resistance in P. aeruginosa keratitis isolates.