Identification of somatic variants in cancer by high-throughput sequencing has become common clinical practice largely because many of these variants may be predictive biomarkers for targeted therapies. However, there can be high sample quality control (QC) failure rates for some tests preventing the return of results. SLIMamp is a patented technology that has been incorporated into commercially available cancer NGS testing kits with the claimed advantage that these kits can interrogate challenging formalin-fixed paraffin-embedded tissue (FFPET) samples with low tumor purity, poor DNA quality, and/or low input DNA, resulting in a high sample QC pass rate. The aim of this study was to substantiate that claim using Pillar's oncoReveal Solid Tumor Panel. 48 samples that had failed one or more pre-analytical QC sample parameters for whole exome sequencing (WES) from ATGC's ISO15189 accredited diagnostic genomics laboratory were acquired. XING Genomic Services (XGS) performed an exploratory data analysis to characterize the samples and then tested the samples in their ISO15189 accredited laboratory. Clinical reports could be generated for 37 samples (77%), of which 29 (60%) contained clinically actionable or significant variants that would not have otherwise been identified. 11 samples were deemed to be unreportable and the sequencing data were likely dominated by artefacts. A novel post-sequencing QC metric was developed which can discriminate between clinically reportable and unreportable samples.

Current guidelines for patients with Chronic Lymphocytic Leukemia (CLL) recommend mutation status determination of the clonotypic IGHV gene prior to treatment initiation in order to guide the choice of the first-line therapy. Currently, commercially available next-generation sequencing (NGS) solutions have technical constraints, as they necessitate at least a 2x300 bp sequencing, which restricts their use for routine practice. The cost of the commercial kits also represents an important drawback. We present a new method called Next-CLL, a ready-to-use strategy to evaluate IGHV gene mutation status using any NGS device (including 2 x 150 bp sequencers) in routine diagnostic laboratories. We validated the performance of Next-CLL on genomic DNA and complementary DNA obtained from 80 CLL patients at diagnosis. Next-CLL identified a productive clone in 100% of cases, whereas PCR with Sanger sequencing led to a 12.5% failure rate. Next-CLL had 100% concordance with the reference technique for IGHV gene identification and allowed assessment of the IGHV mutation status from the leader sequence, following international guidelines. By comparing a large retrospective series of samples, analyzed using Sanger sequencing (n=773) or Next-CLL (n=352), we showed no bias in IGHV usage or mutational status, further validating our strategy in the real-life setting. Next-CLL represents a straight-forward workflow for IGHV analysis in routine practice to assess clonal architecture and prognosis of CLL patients.

The standard-of-care diagnostic prenatal testing includes a combination of cytogenetic methods, such as karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA), using either direct or cultured amniocytes or chorionic villi sampling. However, each technology has its limitations: karyotyping has a low resolution (>5 Mb), FISH is targeted, and CMA does not detect balanced structural variations (SVs). These limitations necessitate the use of multiple tests, either simultaneously or sequentially, to reach a genetic diagnosis. Optical genome mapping (OGM) is an emerging technology that can detect several classes of SVs in a single assay, but it has not been evaluated in the prenatal setting. This validation study analyzed 114 samples that were received in our laboratory for traditional cytogenetic analysis with karyotyping, FISH, and/or CMA. OGM was 100% concordant in identifying the 101 aberrations that included 29 interstitial/terminal deletions, 28 duplications, 26 aneuploidies, 6 absence of heterozygosity regions, 3 triploid genomes, 4 isochromosomes, and 1 translocation; and the method revealed the identity of 3 marker chromosomes and 1 chromosome with additional material not determined by karyotyping. In addition, OGM detected 64 additional clinically reportable SVs in 43 samples. OGM has a standardized laboratory workflow and reporting solution that can be adopted in routine clinical laboratories and demonstrates the potential to replace the current standard-of-care methods for prenatal diagnostic testing.

The coronavirus disease 2019 (COVID-19) pandemic has provided a stage to illustrate that there is considerable value in obtaining rapid, whole-genome-based information about pathogens. This article describes the utility of a commercially available, automated severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2 library preparation; genome sequencing; and a bioinformatics analysis pipeline to provide rapid, near-real-time SARS-CoV-2 variant description. This study evaluated the turnaround time, accuracy, and other quality-related parameters obtained from commercially available automated sequencing instrumentation, from analysis of continuous clinical samples obtained from January 1, 2021, to October 6, 2021. This analysis included a base-by-base assessment of sequencing accuracy at every position in the SARS-CoV-2 chromosome using two commercially available methods. Mean turnaround time, from the receipt of a specimen for SARS-CoV-2 testing to the availability of the results, with lineage assignment, was <3 days. Accuracy of sequencing by one method was 100%, although certain sites on the genome were found repeatedly to have been sequenced with varying degrees of read error rate.

Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency presents with neonatal cholestasis and liver failure in early infancy and features high levels of 3-oxo-Δ4-bile acids in urine. Genetic analysis is needed for definitive diagnosis, because in the neonatal period it can be difficult to distinguish a primary from a secondary enzyme deficiency. By re-analysis of the gene-sequencing data, one AKR1D1 noncanonical splice-site variant (NM_005989.4: c.580-13T>A) with controversial pathogenicity was discovered to be enriched in eight families with clinical and biochemically confirmed AKR1D1 deficiency. Further RNA sequencing of liver tissue suggested this variant causes complete degradation of mRNA. An in vitro minigene experiment indicated that this variant led to partial intron retention or exon jumping, which then leads to coding sequence frameshift and nonsense-mediated mRNA decay. Thus, AKR1D1 variant c.580-13T>A was considered pathogenic and, therefore, should be screened during genetic studies in infants with a suspicion of a congenital bile acid synthetic disorder.

Epithelial ovarian cancers (EOCs) harboring germline or somatic pathogenic variants in BRCA1 and BRCA2 genes show sensitivity to poly(ADP-ribose) polymerase inhibition. It has been suggested that BRCA1 promoter methylation is perhaps a better determinant of therapy response, because of its intrinsic dynamic feature, with respect to genomic scars or gene mutation. Conflicting evidence was reported so far, and the lack of a validated assay to measure promoter methylation was considered a main confounding factor in data interpretation. To contribute to the validation process of a pyrosequencing assay for BRCA1 promoter methylation, 109 EOCs from two Italian centers were reciprocally blindly investigated. By comparing two different pyrosequencing assays, addressing a partially overlapping region of BRCA1 promoter, an almost complete concordance of results was obtained. Moreover, the clinical relevance of this approach was also supported by the finding of BRCA1 transcript down-regulation in BRCA1-methylated EOCs. These findings could lead to the development of a simple and cheap pyrosequencing assay for diagnostics, easily applicable to formalin-fixed, paraffin-embedded tissues. This technique may be implemented in routine clinical practice in the near future to identify EOCs sensitive to poly(ADP-ribose) polymerase inhibitor therapy, thus increasing the subset of women affected by EOCs who could benefit from such treatment.