Automated Identification of Germline de novo Mutations in Family Trios: A Consensus-Based Informatic Approach.

Accurate identification of germline de novo variants (DNVs) remains a challenging problem despite rapid advances in sequencing technologies as well as methods for the analysis of the data they generate, with putative solutions often involving ad hoc filters and visual inspection of identified variants. Here, we present a purely informatic method for the identification of DNVs by analyzing short-read genome sequencing data from proband-parent trios. Our method evaluates variant calls generated by three genome sequence analysis pipelines utilizing different algorithms-GATK HaplotypeCaller, DeepTrio and Velsera GRAF-exploring the assumption that a requirement of consensus can serve as an effective filter for high-quality DNVs. We assessed the efficacy of our method by testing DNVs identified using a previously established, highly accurate classification procedure that partially relied on manual inspection and used Sanger sequencing to validate a DNV subset comprising less confident calls. The results show that our method is highly precise and that applying a force-calling procedure to putative variants further removes false-positive calls, increasing precision of the workflow to 99.6%. Our method also identified novel DNVs, 87% of which were validated, indicating it offers a higher recall rate without compromising accuracy. We have implemented this method as an automated bioinformatics workflow suitable for large-scale analyses without need for manual intervention.

Influence of clusterin genetic variants on IOP elevation in pseudoexfoliation syndrome and pseudoexfoliative glaucoma in Turkish population.

PURPOSE: Pseudoexfoliation syndrome (PEX) is distinguished by the deposition of fibrillary material within the aqueous humor and, in most cases, causes pseudoexfoliative glaucoma (PEG). The pathophysiologies of PEX and PEG are not completely explained. Therefore, this study aimed to assess the potential relationship between single nucleotide polymorphisms (SNPs) in the 3' untranslated region or introns of the clusterin gene (CLU) and the susceptibility to developing PEG or PEX. METHODS: Two hundred and forty patients with PEX, 239 patients with PEG, and 240 control subjects were included. Genotyping was carried out using real-time PCR (rs2279590 C/T and rs1532278 C/T) or PCR followed by restriction endonuclease digestion (rs11136000 C/T and rs3087554 T/C). RESULTS: The minor alleles or genotypes of CLU SNPs were not significantly associated with PEX or PEG. IOP values of patients with PEX carrying the homozygote polymorphic TT genotype were significantly elevated compared with PEX cases with the CT or CC genotypes for rs2279590, rs11136000 and rs1532278 (P = .009, P = .007, P = .010, respectively). CONCLUSION: We present the first evidence that three SNPs in CLU gene (rs2279590, rs11136000 and rs1532278) might induce a rise in IOP in patients with PEX, conferring susceptibility to develop PEG.

Pan-African genome demonstrates how population-specific genome graphs improve high-throughput sequencing data analysis.

Graph-based genome reference representations have seen significant development, motivated by the inadequacy of the current human genome reference to represent the diverse genetic information from different human populations and its inability to maintain the same level of accuracy for non-European ancestries. While there have been many efforts to develop computationally efficient graph-based toolkits for NGS read alignment and variant calling, methods to curate genomic variants and subsequently construct genome graphs remain an understudied problem that inevitably determines the effectiveness of the overall bioinformatics pipeline. In this study, we discuss obstacles encountered during graph construction and propose methods for sample selection based on population diversity, graph augmentation with structural variants and resolution of graph reference ambiguity caused by information overload. Moreover, we present the case for iteratively augmenting tailored genome graphs for targeted populations and demonstrate this approach on the whole-genome samples of African ancestry. Our results show that population-specific graphs, as more representative alternatives to linear or generic graph references, can achieve significantly lower read mapping errors and enhanced variant calling sensitivity, in addition to providing the improvements of joint variant calling without the need of computationally intensive post-processing steps.

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions.

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

Evaluation of Tear Fluid and Aqueous Humor Concentration of Clusterin as Biomarkers for Early Diagnosis of Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma.

PURPOSE: Pseudoexfoliation syndrome (PEX) is an age-related disorder of the extracellular matrix characterized by the accumulation of fibrillary deposits in the anterior chamber of the eye, which leads to the development of pseudoexfoliative glaucoma (PEG). Early identification of subjects with higher susceptibility to PEX and PEG development is very important so that these conditions are managed at earlier stages, which requires that an objective biomarker is defined. Therefore, in the present study, we aimed to determine if aqueous humor and tear fluid concentrations of clusterin, an extracellular chaperone, are objective biomarkers for PEX and PEG risk. METHODS: Tear fluid was obtained from 80 patients with PEG, 80 patients with PEX, and 80 controls, using Schirmer strips. Aqueous humor was also collected during cataract surgery from 12 patients with PEG, 17 patients with PEX, and 22 controls, who also gave tear samples. Clusterin concentration was determined by ELISA. RESULTS: Clusterin concentration in aqueous humor was significantly higher in patients with PEG than in PEX cases (P = .002) and controls (P = .004). Receiver operating characteristics analysis revealed that this parameter is a robust classifier to distinguish PEG and PEX cases. Tear fluid clusterin concentrations did not differ significantly between groups. Aqueous humor and tear fluid levels of clusterin were not significantly correlated. CONCLUSIONS: In conclusion, tear fluid clusterin level in patients with PEG and PEX was determined for the first time, which showed no difference between study groups. Aqueous humor clusterin level was markedly higher in patients with PEG.

Paraoxonase 1 (PON1) promoter (-107T/C) and coding region (192Q/R and 55L/M) genetic variations in pseudoexfoliation syndrome and pseudoexfoliative glaucoma risk.

PURPOSE: Pseudoexfoliation syndrome (PEX) is characterized by the accumulation of microscopic extracellular material in the anterior chamber of the eye and can lead to the development of pseudoexfoliative glaucoma (PEG) in some patients. The pathogenesis of PEX is not fully understood, and there are no objective biomarkers for its early diagnosis. Recent research has indicated that oxidative stress and inflammation might play a role in the pathophysiology of the production of pseudoexfoliation material. Therefore, in the present study, we aimed to analyze the possible association between three genetic variants of paraoxonase 1 (PON1), a well-recognized antioxidant and anti-inflammatory enzyme, and PEX/PEG. METHODS: The study population consisted of patients with PEX (n = 150), patients with PEG (n = 150), and control subjects (n = 150). PON1 -107T/C, 192Q/R, and 55L/M genotypes were determined using PCR followed by restriction fragment length polymorphism analysis. The correlation between these genetic alterations and clinical visual characteristics was also investigated. RESULTS: The minor allele frequencies and genotype distributions of PON1 did not differ significantly between the PEG, PEX, and control groups. Moreover, PON1 genotypes did not significantly influence visual clinical parameters in stratification analysis. On the other hand, in correlation analysis, pattern standard deviation was significantly correlated with the -107T/C genotypes in PEX group. In addition, intraocular pressure was correlated with the 55L/M genotypes and mean deviation was correlated with the -107T/C genotypes in the control group. Furthermore, intraocular pressure was significantly inversely correlated with sex (r =  - 0.116, P = 0.011) in the overall study group. Logistic regression analysis showed that having a PON1 -107TC or CC genotype is significantly associated with PEX (OR = 1.909, P = 0.020). CONCLUSIONS: This study, for the first time, analyzed the relationship between PON1 genetic variants, clinical visual parameters, and PEX/PEG. The results indicated a possible role for the PON1 promoter variant in PEX.