Familial co-segregation and the emerging role of long-read sequencing to re-classify variants of uncertain significance in inherited retinal diseases.

Phasing genetic variants is essential in determining those that are potentially disease-causing. In autosomal recessive inherited retinal diseases (IRDs), reclassification of variants of uncertain significance (VUS) can provide a genetic diagnosis in indeterminate compound heterozygote cases. We report four cases in which familial co-segregation demonstrated a VUS resided in trans to a known pathogenic variant, which in concert with other supporting criteria, led to the reclassification of the VUS to likely pathogenic, thereby providing a genetic diagnosis in each case. We also demonstrate in a simplex patient without access to family members for co-segregation analysis that targeted long-read sequencing can provide haplotagged variant calling. This can elucidate if variants reside in trans and provide phase of genetic variants from the proband alone without parental testing. This emerging method can alleviate the bottleneck of haplotype analysis in cases where genetic testing of family members is unfeasible to provide a complete genetic diagnosis.

Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients.

Inherited retinal degenerations (IRDs) are a heterogeneous group of predominantly monogenic disorders with over 300 causative genes identified. Short-read exome sequencing is commonly used to genotypically diagnose patients with clinical features of IRDs, however, in up to 30% of patients with autosomal recessive IRDs, one or no disease-causing variants are identified. Furthermore, chromosomal maps cannot be reconstructed for allelic variant discovery with short-reads. Long-read genome sequencing can provide complete coverage of disease loci and a targeted approach can focus sequencing bandwidth to a genomic region of interest to provide increased depth and haplotype reconstruction to uncover cases of missing heritability. We demonstrate that targeted adaptive long-read sequencing on the Oxford Nanopore Technologies (ONT) platform of the USH2A gene from three probands in a family with the most common cause of the syndromic IRD, Usher Syndrome, resulted in greater than 12-fold target gene sequencing enrichment on average. This focused depth of sequencing allowed for haplotype reconstruction and phased variant identification. We further show that variants obtained from the haplotype-aware genotyping pipeline can be heuristically ranked to focus on potential pathogenic candidates without a priori knowledge of the disease-causing variants. Moreover, consideration of the variants unique to targeted long-read sequencing that are not covered by short-read technology demonstrated higher precision and F1 scores for variant discovery by long-read sequencing. This work establishes that targeted adaptive long-read sequencing can generate targeted, chromosome-phased data sets for identification of coding and non-coding disease-causing alleles in IRDs and can be applicable to other Mendelian diseases.

Machine Learning Prediction of Adenovirus D8 Conjunctivitis Complications from Viral Whole-Genome Sequence.

Objective: To obtain complete DNA sequences of adenoviral (AdV) D8 genome from patients with conjunctivitis and determine the relation of sequence variation to clinical outcomes. Design: This study is a post hoc analysis of banked conjunctival swab samples from the BAYnovation Study, a previously conducted, randomized controlled clinical trial for AdV conjunctivitis. Participants: Ninety-six patients with AdV D8-positive conjunctivitis who received placebo treatment in the BAYnovation Study were included in the study. Methods: DNA from conjunctival swabs was purified and subjected to whole-genome viral DNA sequencing. Adenovirus D8 variants were identified and correlated with clinical outcomes, including 2 machine learning methods. Main Outcome Measures: Viral DNA sequence and development of subepithelial infiltrates (SEIs) were the main outcome measures. Results: From initial sequencing of 80 AdV D8-positive samples, full adenoviral genome reconstructions were obtained for 71. A total of 630 single-nucleotide variants were identified, including 156 missense mutations. Sequence clustering revealed 3 previously unappreciated viral clades within the AdV D8 type. The likelihood of SEI development differed significantly between clades, ranging from 83% for Clade 1 to 46% for Clade 3. Genome-wide analysis of viral single-nucleotide polymorphisms failed to identify single-gene determinants of outcome. Two machine learning models were independently trained to predict clinical outcome using polymorphic sequences. Both machine learning models correctly predicted development of SEI outcomes in a newly sequenced validation set of 16 cases (P = 1.5 × 10-5). Prediction was dependent on ensemble groups of polymorphisms across multiple genes. Conclusions: Adenovirus D8 has ≥ 3 prevalent molecular substrains, which differ in propensity to result in SEIs. Development of SEIs can be accurately predicted from knowledge of full viral sequence. These results suggest that development of SEIs in AdV D8 conjunctivitis is largely attributable to pathologic viral sequence variants within the D8 type and establishes machine learning paradigms as a powerful technique for understanding viral pathogenicity.

Targeted long-read sequencing allows for rapid identification of pathogenic disease-causing variants in retinoblastoma.

BACKGROUND: Identification of disease-causing variants of the retinoblastoma gene (RB1), the predominant cause of retinoblastoma, is challenging. Targeted long-read genome sequencing offers a novel approach to resolve the diverse range of pathogenic variants in RB1 and provides haplotype information rapidly. MATERIALS AND METHODS: Genomic DNA was isolated from a venipuncture blood draw of a retinoblastoma patient. Whole genome sequencing (WGS) was carried out using the short-read Ilumina platform. WGS and targeted sequencing of RB1 was accomplished using the long-read Oxford Nanopore Technologies (ONT) platform. Deep-learning frameworks allowed haplotagging, variant calling, and variant annotation of both short- and long-read data. RESULTS: Targeted long-read sequencing of the RB1 gene allowed for enhanced depth of read coverage for discovery of rare variants and haplotype analysis. A duplication leading to a frameshift and early termination in RB1 was identified as the most deleterious variant by all sequencing methods, with long-read technology providing additional information of methylation signal and haplotype information. More importantly, there was greater than 98% concordance of RB1 variants identified between short-read and targeted long-read sequencing modalities. CONCLUSIONS: Targeted long-read technology allows for focused sequencing effort for variant discovery. Application of this for the first time in a retinoblastoma patient allowed haplotagged variant identification and demonstrated excellent concordance with benchmark short-read sequencing. The added benefit of targeted long-read sequencing to resolve disease-causing genomic variation in RB1 rapidly from a blood draw will provide a more definitive diagnosis of heritable RB and guide management decisions for patients and their families.

Deep Metagenomic Sequencing for Endophthalmitis Pathogen Detection Using a Nanopore Platform.

PURPOSE: To evaluate the utility of nanopore sequencing for identifying potential causative pathogens in endophthalmitis, comparing culture results against full-length 16S rRNA nanopore sequencing (16S Nanopore), whole genome nanopore sequencing (Nanopore WGS), and Illumina (Illumina WGS). DESIGN: Cross-sectional diagnostic comparison. METHODS: Patients with clinically suspected endophthalmitis underwent intraocular vitreous biopsy as per standard care. Clinical samples were cultured by conventional methods, together with full-length 16S rRNA and WGS using nanopore and Illumina sequencing platforms. RESULTS: Of 23 patients (median age 68.5 years [range 47-88]; 14 males [61%]), 18 cases were culture-positive. Nanopore sequencing identified the same cultured organism in all of the culture-positive cases and identified potential pathogens in two culture-negative cases (40%). Nanopore WGS was able to additionally detect the presence of bacteriophages in three samples. The agreements at genus level between culture and 16S Nanopore, Nanopore WGS, and Illumina WGS were 75%, 100%, and 78%, respectively. CONCLUSIONS: Whole genome sequencing has higher sensitivity and provides a viable alternative to culture and 16S sequencing for detecting potential pathogens in endophthalmitis. Moreover, WGS has the ability to detect other potential pathogens in culture-negative cases. Whilst Nanopore and Illumina WGS provide comparable data, nanopore sequencing provides potential for cost-effective point-of-care diagnostics.

Molecular and Clinical Characterization of Human Adenovirus E4-Associated Conjunctivitis.

PURPOSE: To determine the characteristics of conjunctivitis associated with human adenovirus E4 (AdV E4). METHODS: Samples and outcomes from 500 patients with conjunctivitis were obtained from the NVC-422 randomized controlled clinical trial comparing auriclosene to placebo. Molecular typing identified 36 cases associated with AdV E4. Signs and symptoms at presentation and at the day 18 endpoint were compared with the larger cohort of 262 subjects with conjunctivitis caused by due to AdV D8. Full viral genomes of 22 AdV E4 isolates were reconstructed. RESULTS: AdV E4 was the most frequently identified adenoviral type in conjunctivitis cases from the United States. Signs and symptoms at presentation were comparable to those associated with AdV D8. Viral load at presentation was comparable between groups but resolution was more rapid in the AdV E4 group. Clinical signs were fully resolved by day 18 in 26 of 36 (72%) patients with AdV E4. Subepithelial infiltrates developed in 12 of 36 (33%) patients with AdV E4 compared with 98 of 215 (45%) patients with AdV D8 (P = .0001). One hundred twenty-four polymorphisms were observed among 22 whole viral genome sequences, which clustered into 3 clades. Patients in each clade developed subepithelial infiltrates. Neither single nucleotide polymorphism analysis nor machine learning approaches identified specific sequence features predictive of presenting signs or outcome. CONCLUSIONS: AdV E4 conjunctivitis may be indistinguishable at presentation from AdV D8-associated disease. Resolution of viral load for AdV E4 appears more rapid than for AdV D8, and the risk for subepithelial infiltrates appears lower. Multiple substrains of AdV E4 are in circulation but all appeared equivalently pathogenic for conjunctivitis. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Hospitalization and mortality associated with SARS-CoV-2 viral clades in COVID-19.

The COVID-19 epidemic of 2019-20 is due to the novel coronavirus SARS-CoV-2. Following first case description in December, 2019 this virus has infected over 10 million individuals and resulted in at least 500,000 deaths world-wide. The virus is undergoing rapid mutation, with two major clades of sequence variants emerging. This study sought to determine whether SARS-CoV-2 sequence variants are associated with differing outcomes among COVID-19 patients in a single medical system. Whole genome SARS-CoV-2 RNA sequence was obtained from isolates collected from patients registered in the University of Washington Medicine health system between March 1 and April 15, 2020. Demographic and baseline clinical characteristics of patients and their outcome data including their hospitalization and death were collected. Statistical and machine learning models were applied to determine if viral genetic variants were associated with specific outcomes of hospitalization or death. Full length SARS-CoV-2 sequence was obtained 190 subjects with clinical outcome data. 35 (18.4%) were hospitalized and 14 (7.4%) died from complications of infection. A total of 289 single nucleotide variants were identified. Clustering methods demonstrated two major viral clades, which could be readily distinguished by 12 polymorphisms in 5 genes. A trend toward higher rates of hospitalization of patients with Clade 2 infections was observed (p = 0.06, Fisher's exact). Machine learning models utilizing patient demographics and co-morbidities achieved area-under-the-curve (AUC) values of 0.93 for predicting hospitalization. Addition of viral clade or sequence information did not significantly improve models for outcome prediction. In summary, SARS-CoV-2 shows substantial sequence diversity in a community-based sample. Two dominant clades of virus are in circulation. Among patients sufficiently ill to warrant testing for virus, no significant difference in outcomes of hospitalization or death could be discerned between clades in this sample. Major risk factors for hospitalization and death for either major clade of virus include patient age and comorbid conditions.

Outcomes associated with SARS-CoV-2 viral clades in COVID-19.

Background The COVID-19 epidemic of 2019-20 is due to the novel coronavirus SARS-CoV-2. Following first case description in December, 2019 this virus has infected over 10 million individuals and resulted in at least 500,000 deaths world-wide. The virus is undergoing rapid mutation, with two major clades of sequence variants emerging. This study sought to determine whether SARS-CoV-2 sequence variants are associated with differing outcomes among COVID-19 patients in a single medical system. Methods Whole genome SARS-CoV-2 RNA sequence was obtained from isolates collected from patients registered in the University of Washington Medicine health system between March 1 and April 15, 2020. Demographic and baseline medical data along with outcomes of hospitalization and death were collected. Statistical and machine learning models were applied to determine if viral genetic variants were associated with specific outcomes of hospitalization or death. Findings Full length SARS-CoV-2 sequence was obtained 190 subjects with clinical outcome data. 35 (18.4%) were hospitalized and 14 (7.4%) died from complications of infection. A total of 289 single nucleotide variants were identified. Clustering methods demonstrated two major viral clades, which could be readily distinguished by 12 polymorphisms in 5 genes. A trend toward higher rates of hospitalization of patients with Clade 2 was observed (p=0.06). Machine learning models utilizing patient demographics and co-morbidities achieved area-under-the-curve (AUC) values of 0.93 for predicting hospitalization. Addition of viral clade or sequence information did not significantly improve models for outcome prediction. Conclusion SARS-CoV-2 shows substantial sequence diversity in a community-based sample. Two dominant clades of virus are in circulation. Among patients sufficiently ill to warrant testing for virus, no significant difference in outcomes of hospitalization or death could be discerned between clades in this sample. Major risk factors for hospitalization and death for either major clade of virus include patient age and comorbid conditions.

Prognostic Utility of Whole-Genome Sequencing and Polymerase Chain Reaction Tests of Ocular Fluids in Postprocedural Endophthalmitis.

PURPOSE: To associate detection of potential pathogen DNA in endophthalmitis with clinical outcomes. DESIGN: Prospective cohort study. METHODS: Patients in whom endophthalmitis was diagnosed following an intraocular procedure were recruited. Clinical outcome data from baseline, week-1, month-1, and month-3 visits were collected. Intraocular biopsy samples were cultured by standard methods. Quantitative polymerase chain reaction (qPCR) was performed for specific pathogens and whole-genome sequencing (WGS). RESULTS: A total of 50 patients (mean age 72 years old; 52% male) were enrolled. Twenty-four cases were culture-positive and 26 were culture-negative. WGS identified the cultured organism in 76% of culture-positive cases and identified potential pathogens in 33% of culture-negative cases. Month-1 and -3 visual acuities did not vary by pathogen-positive versus pathogen-negative cases as detected by either culture or WGS. Visual outcomes of Staphylococcus epidermidis endophthalmitis were no different than those of pathogen-negative cases, whereas the patients infected with other pathogens showed worse outcome. Higher baseline bacterial DNA loads of bacteria other than those of S epidermidis detected by WGS were associated with worse month-1 and -3 visual acuity, whereas the S epidermidis loads did not appear to influence outcomes. Torque teno virus (TTV) and Merkel cell polyomavirus (MCV) were detected by qPCR in 49% and 19% of cases, respectively. Presence of TTV at presentation was associated with a higher rate of secondary pars plana vitrectomy (P = .009) and retinal detachment (P = .022). CONCLUSIONS: The presence and higher load of bacteria other than S epidermidis detected by WGS or DNA from TTV by qPCR in ocular fluids is associated with worse outcomes in post-procedure endophthalmitis.