Blommel, Joseph H.

Abstract Background Massively parallel sequencing (MPS) of nucleic acids has been a transformative technology for basic and applied genomic science, increasing efficiencies and decreasing costs to enable studies of unprecedented scope and impact. In clinical settings, these technological and scientific advances have led to the development of tests that are increasingly fast, comprehensive, and more frequently employed. Practitioners of genomic medicine have applied these tools across clinical settings, including diagnosis of inherited disorders and cancers and infectious disease detection and surveillance. In recent years, the commercial marketplace for MPS sequencers and reagents has been dominated by a few companies. The growing demand for sequencing has led to the recent emergence of several new sequencing platforms with techniques that may provide alternatives or improvements to existing workflows or allow the adoption of sequencing workflows in new settings. Clinical genomics laboratories will evaluate these platforms from a unique perspective, focusing on how technological advancements can improve patient care. Content This review describes short-read sequencing platforms provided by Illumina, Element Biosciences, MGI, PacBio, Singular Genomics, Thermo Fisher Scientific, and Ultima Genomics. This review discusses their innovative approaches, principles, workflows, and applications. Summary This review aims to inform laboratory geneticists, clinicians, and researchers about emerging short-read technologies and their applications in clinical genomics. By highlighting their principles and potential contributions, we aim to assist laboratories in selecting suitable solutions for their sequencing needs considering key factors such as applications, throughput, and integration with existing laboratory workflows.

Abstract Background The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal. Methods Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability. Results We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices. Conclusions This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing.

Abstract Background We evaluated the analytical sensitivity and specificity of 4 rapid antigen diagnostic tests (Ag RDTs) for severe acute respiratory syndrome coronavirus 2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. Methods Three hundred fifty (150 negative and 200 RT-qPCR positive) residual PBS samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR–positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR–positive, antigen-positive samples for further analysis. Results All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1 500 000 copies/mL RNA, and detected ≥75% of samples with viral load of 500 000 to 1 500 000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50 000 to 500 000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load <50 000 copies/mL compared to the BD Veritor. Among samples with detectable antigen and viral load <50 000 copies/mL, sensitivity of the Ag RDT varied between 13.0% (BD Veritor) and 78.3% (ACON FIA). Conclusions Ag RDTs differ significantly in analytical sensitivity, particularly at viral load <500 000 copies/mL.

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations. Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed. Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon). Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis. Disclosures Jen: Celgene: Employment.