Genome sequencing in microfabricated high-density picolitre reactors.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Investigation of Direct-Acting Oral Anticoagulants and the Incidence of Venous Thromboembolism in Patients Weighing ≥120 kg Compared to Patients Weighing <120 kg.

BACKGROUND: Direct oral anticoagulants (DOACs) present a favorable alternative to warfarin based on the decreased burden of monitoring and fewer drug and food interactions. Although studied in the general population, limited clinical data justifying efficacy in patients weighing ≥120 kg present concern for using DOACs in this specific population. OBJECTIVE: The purpose was to identify if a difference exists in incidence of recurrent thromboembolic events in patients receiving a DOAC for the indication of venous thromboembolism (VTE) weighing ≥120 kg compared to patients weighing <120 kg. METHODS: A retrospective database analysis was conducted with patients on apixaban, dabigatran, or rivaroxaban for treatment of VTE from the Veterans Integrated Service Network 8 between January 2012 and June 2017. The primary outcome was incidence of recurrent VTEs while on anticoagulation. Fisher's exact tests were used to evaluate difference in VTEs between the groups. RESULTS: There were 133 patients weighing ≥120 kg and 1063 patients weighing <120 kg identified within the 5-year time frame that met inclusion criteria. Although no statistically significant difference was found in incidence of recurrent VTEs between study groups (0.8% vs 1.1%; odds ratio: 0.66; 95% confidence interval: 0.09-5.14; P = .69) few events occurred limiting the power to be able to detect a difference. CONCLUSION: This study found no difference in VTE recurrence in patients weighing ≥120 kg compared to patients <120 kg with few events in either group. Although promising, additional studies are needed to confirm these findings.

A Streamlined Approach to Prader-Willi and Angelman Syndrome Molecular Diagnostics.

Establishing or ruling out a molecular diagnosis of Prader-Willi or Angelman syndrome (PWS/AS) presents unique challenges due to the variety of different genetic alterations that can lead to these conditions. Point mutations, copy number changes, uniparental isodisomy (i-UPD) 15 of two subclasses (segmental or total isodisomy), uniparental heterodisomy (h-UPD), and defects in the chromosome 15 imprinting center can all cause PWS/AS. Here, we outline a combined approach using whole-exome sequencing (WES) and DNA methylation data with methylation-sensitive multiplex ligation-dependent probe amplification (MLPA) to establish both the disease diagnosis and the mechanism of disease with high sensitivity using current standard of care technology and improved efficiency compared to serial methods. The authors encourage the use of this approach in the clinical setting to confirm and establish the diagnosis and genetic defect which may account for the secondary genetic conditions that may be seen in those with isodisomy 15, impacting surveillance and counseling with more accurate recurrence risks. Other similarly affected individuals due to other gene disorders or cytogenetic anomalies such as Rett syndrome or microdeletions would also be identified with this streamlined approach.

The Value of Parental Testing by Next-Generation Sequencing Includes the Detection of Germline Mosaicism.

When a potential disease-causing variant is detected in a proband, parental testing is used to determine the mode of inheritance. This study demonstrates that next-generation sequencing (NGS) is uniquely well suited for parental testing, in particular because of its ability to detect clinically relevant germline mosaicism. Parental variant testing by NGS was performed in a clinical laboratory for 1 year. The detection of mosaicism by NGS was compared with its detection by Sanger sequencing. Eight cases of previously unrevealed mosaicism were detected by NGS across eight different genes. Mosaic variants were differentiated from sequencing noise using custom bioinformatics analyses in combination with familial inheritance data and complementary Sanger sequencing. Sanger sequencing detected mosaic variants with allele fractions ≥8% by NGS, but could not detect mosaic variants below that level. Detection of germline mosaicism by NGS is invaluable to parents, providing a more accurate recurrence risk that can alter decisions on family planning and pregnancy management. Because NGS can also confirm parentage and increase scalability, it simultaneously streamlines and strengthens the variant curation process. These features make NGS the ideal method for parental testing, superior even to Sanger sequencing for most genomic loci.

P2Y12 inhibitors: do they increase cancer risk?

Treatment with dual antiplatelet therapy (DAPT), typically combining a P2Y12 inhibitor with aspirin, is the standard of care for the prevention of coronary stent thrombosis, especially post revascularization and in the setting of acute coronary syndromes (ACS). Determining the appropriate duration has been debated as prolonged courses have been associated with reduced thrombotic complications. Despite proven benefit, there have been reports of a potential cancer risk associated with DAPT following the FDA's review of the TRITON-TIMI 38 trial and the DAPT trial. The latter revealed an increased risk of non-cardiovascular death, which was driven by more bleeding and cancer-related deaths. This further clouds the decision if longer courses of DAPT should be recommended. Several trials and meta-analyses have been conducted to further review this cancer risk with P2Y12 inhibitors. This manuscript intends to evaluate current literature to determine if there is a risk of cancer for patients on DAPT and its consequences in the management of cardiovascular disease.

From WOEST to AUGUSTUS: a review of safety and efficacy of triple versus dual antithrombotic regimens in patients with atrial fibrillation requiring percutaneous coronary intervention for acute coronary syndrome.

For patients with atrial fibrillation with concomitant acute coronary syndrome (ACS) requiring percutaneous coronary intervention (PCI), the increased risk of bleeding associated with the use triple therapy is well established. However, there is question whether it is a necessary risk for patients to prevent stroke and stent thrombosis. The purpose of this article is to highlight the findings of prior studies evaluating the comparative safety and efficacy of dual and triple antithrombotic regimens in the subgroup of atrial fibrillation patients requiring PCI for ACS. Trials that evaluated dual versus triple antithrombotic therapy demonstrated post-PCI treatment with a P2Y12 inhibitor alone was safer than aspirin plus a P2Y12 inhibitor in patients also taking an anticoagulant for atrial fibrillation. Data regarding ischemic outcomes have not suggested harm with the omission of aspirin, but these studies have not been powered to assess efficacy outcomes, especially in ACS patients. These studies also demonstrate a significant reduction in bleeding events when aspirin is excluded from the post-PCI regimen in the ACS subgroup of atrial fibrillation patients. Further studies, with added focus on the ACS subgroup, are needed to potentially confirm that dual therapy may be as efficacious as triple therapy in ACS patients with atrial fibrillation.