Validation Of Whole Genome Sequencing Research Articles

High Concordance between the Whole Genome Sequencing data Analysis Methods for Vibrio Cholerae Surveillance in Finland and Norway

High Concordance between the Whole Genome Sequencing data Analysis Methods for Vibrio Cholerae Surveillance in Finland and Norway

The Brazilian Rare Genomes Project: Validation of Whole Genome Sequencing for Rare Diseases Diagnosis.

Rare diseases affect up to 13.2 million individuals in Brazil. The Brazilian Rare Genomes Project is envisioned to further the implementation of genomic medicine into the Brazilian public healthcare system. Here we report the validation results of a whole genome sequencing (WGS) procedure for implementation in clinical laboratories. In addition, we report data quality for the first 1,200 real-world patients sequenced. We sequenced a well-characterized group of 76 samples, including seven gold standard genomes, using a PCR-free WGS protocol on Illumina Novaseq 6,000 equipment. We compared the observed variant calls with their expected calls, observing good concordance for single nucleotide variants (SNVs; mean F-measure = 99.82%) and indels (mean F-measure = 99.57%). Copy number variants and structural variants events detection performances were as expected (F-measures 96.6% and 90.3%, respectively). Our WGS protocol presented excellent intra-assay reproducibility (coefficients of variation ranging between 0.03% and 0.20%) and inter-assay reproducibility (coefficients of variation ranging between 0.02% and 0.09%). Limitations of the WGS protocol include the inability to confidently detect variants such as uniparental disomy, balanced translocations, repeat expansion variants, and low-level mosaicism. In summary, the observed performance of the WGS protocol was in accordance with that seen in the best centers worldwide. The Rare Genomes Project is an important initiative to bring pivotal improvements to the quality of life of the affected individuals.

Analysis of Factors Influencing Multidrug-Resistant Tuberculosis and Validation of Whole-Genome Sequencing in Children with Drug-Resistant Tuberculosis.

ObjectivePediatric tuberculosis (TB) is one of the top ten causes of death in children. Our study was to analyze influencing factors of multidrug-resistant tuberculosis (MDR-TB) and validation of whole-genome sequencing (WGS) used in children with drug-resistant TB (DR-TB).MethodsAll Mycobacterium tuberculosis (Mtb) strains were isolated from patients aged below 18 years old of Children’s Hospital of Chongqing Medical University, China. A total of 208 Mtb isolates were tested for eight anti-TB drugs with phenotypic drug susceptibility test (DST) and for genetic prediction of the susceptible profile with WGS. The patients corresponding to each strain were grouped according to drug resistance and genotype. Influencing factors of MDR-TB and DR-TB were analyzed.ResultsAccording to the phenotypic DST and WGS, 82.2% of Mtb strains were susceptible to all eight drugs, and 6.3% were MDR-TB. Using the phenotypic DSTs as the gold standard, the kappa value of WGS to predict isoniazid, rifampin, ethambutol, rifapentine, prothionamide, levofloxacin, moxifloxacin and amikacin was 0.84, 0.89, 0.59, 0.86, 0.89, 0.82, 0.88 and 1.00, respectively. There was significant difference in the distribution of severe TB, diagnosis, treatment and outcome between MDR and drug-susceptible group (P<0.05). The distribution of severe TB and treatment between DR and drug-susceptible group was statistically different (P<0.05). The results of binary logistic regression showed that Calmette–Guérin bacillus (BCG) vaccine is the protective factor for MDR-TB (OR=0.19), and MDR-TB is the risk factor for PTB and EPTB (OR=17.98).ConclusionThe BCG vaccine is a protective factor for MDR-TB, and MDR-TB might not be confined to pulmonary infection, spreading to extrapulmonary organs in children. MDR-TB had more severe cases and a lower recovery rate than drug-susceptible TB. WGS could provide an accurate prediction of drug susceptibility test results for anti-TB drugs, which are needed for the diagnosis and precise treatment of TB in children.

Clinical Validation of Whole Genome Sequencing for Cancer Diagnostics

Whole genome sequencing (WGS) using fresh-frozen tissue and matched blood samples from cancer patients may become the most complete genetic tumor test. With the increasing availability of small biopsies and the need to screen more number of biomarkers, the use of a single all-inclusive test is preferable over multiple consecutive assays. To meet high-quality diagnostics standards, we optimized and clinically validated WGS sample and data processing procedures, resulting in a technical success rate of 95.6% for fresh-frozen samples with sufficient (≥20%) tumor content. Independent validation of identified biomarkers against commonly used diagnostic assays showed a high sensitivity (recall; 98.5%) and precision (positive predictive value; 97.8%) for detection of somatic single-nucleotide variants and insertions and deletions (across 22 genes), and high concordance for detection of gene amplification (97.0%; EGFR and MET) as well as somatic complete loss (100%; CDKN2A/p16). Gene fusion analysis showed a concordance of 91.3% between DNA-based WGS and an orthogonal RNA-based gene fusion assay. Microsatellite (in)stability assessment showed a sensitivity of 100% with a precision of 94%, and virus detection (human papillomavirus), an accuracy of 100% compared with standard testing. In conclusion, whole genome sequencing has a >95% sensitivity and precision compared with routinely used DNA techniques in diagnostics, and all relevant mutation types can be detected reliably in a single assay.

Validation of whole genome sequencing from dried blood spots

BackgroundDried blood spots (DBS) are a relatively inexpensive source of nucleic acids and are easy to collect, transport, and store in large-scale field surveys, especially in resource-limited settings. However, their performance in whole-genome sequencing (WGS) relative to that of venous blood DNA has not been analyzed for various downstream applications.MethodsThis study compares the WGS performance of DBS paired with venous blood samples collected from 12 subjects.ResultsResults of standard quality checks of coverage, base quality, and mapping quality were found to be near identical between DBS and venous blood. Concordance for single-nucleotide variants, insertions and deletions, and copy number variants was high between these two sample types. Additionally, downstream analyses typical of population-based studies were performed, such as mitochondrial heteroplasmy detection, haplotype analysis, mitochondrial copy number changes, and determination of telomere lengths. The absolute mitochondrial copy number values were higher for DBS than for venous blood, though the trend in sample-to-sample variation was similar between DBS and blood. Telomere length estimates in most DBS samples were on par with those from venous blood.ConclusionDBS samples can serve as a robust and feasible alternative to venous blood for studies requiring WGS analysis.

1189O Validation of whole genome sequencing in routine clinical practice

Clinical-grade Whole Genome Sequencing (cWGS) is currently being implemented in routine practice. In the WIDE study (WGS Implementation in standard cancer Diagnostics for Every cancer patient), cWGS is performed on a prospective cohort of 1,200 patients (stage IV solid tumors). Here we report the results on feasibility and clinical validity (primary endpoints) of the first 600 patients. cWGS was conducted independently of, but in parallel with, validated Standard-of-Care (SOC) diagnostics. For 47% of patients, this included SOC molecular diagnostics (MolDx). cWGS and MolDx results were compared and discussed in a dedicated tumor board. Initial discordances were further evaluated by additional tests. cWGS was successfully performed in 69% (414/602) of patients with a technical success rate of 96% (414/433). Ineligibility for cWGS was mostly caused by an insufficient number of tumor cells (<20%) in the received biopsy (86%, 145/169). MolDx was performed in 47% (283/602) and was successful in 95% (267/283) of patients. cWGS showed a median turn-around-time (TAT) of 14 days, which decreased incrementally by continuous improvements to the clinical procedure and cWGS pipeline. In total, 480 genomic biomarkers for clinical validation were identified by SOC MolDx. cWGS showed an error rate of 4.2% (20/480) compared to a SOC MolDx error rate of 1% (5/480). Most of the biomarkers that were not reported by cWGS (19 of 20) were present in the raw data but not reliably identified due to very low variant allele frequencies. Improvements to further increase the sensitivity are currently being implemented with an anticipated reduction of the cWGS error rate to 2%. Overall, cWGS identified a clinically actionable (routine practice and experimental) biomarker in 74% of all patients tested. Compared to SOC MolDx, cWGS identified one or more additional treatment options in 69% (197/287) of patients. Interestingly, in patients that were not tested by SOC MolDx, actionable variants were identified in 60% (76/126). Based on the first 600 patients of the WIDE study, cWGS was found to be clinically feasible in routine molecular diagnostics in a comprehensive cancer center setting and has added value by providing additional treatment options for the majority of patients.

CLIA-compliant validation of Whole Genome Sequencing (WGS) for clinical microbiological applications: experience of one state public health reference laboratory

CLIA-compliant validation of Whole Genome Sequencing (WGS) for clinical microbiological applications: experience of one state public health reference laboratory

Clinical-grade validation of whole genome sequencing reveals robust detection of low-frequency variants and copy number alterations in CLL.

The 100000 Genome Project aims to develop a diagnostics platform by introducing whole genome sequencing (WGS) into clinical practice. Samples from patients with chronic lymphocytic leukaemia were subjected to WGS. WGS detection of single nucleotide variants and insertion/deletions were validated by targeted next generation sequencing showing high concordance (96·3%), also for detection of sub-clonal variants and low-frequency TP53 variants. Copy number alteration detection was verified by fluorescent insitu hybridisation and genome-wide single nucleotide polymorphism array (concordances of 86·7% and 92·9%, respectively), confirming adequate sensitivity by WGS. Our results confirm that WGS can provide comprehensive genomic characterisation for clinical trials, drug discovery and, ultimately, precision medicine.