Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Template Amplicons.

Our goal was to assess the accuracy of next generation sequencing (NGS) compared to Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n=30) were sequenced with Sanger, or re-amplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies [ONT] and Pacific Bioscience [PB]. For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical [n=9] or nearly identical [n=14] compared to Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had 35 ambiguous calls in five amplicons, and 0 ambiguities in two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template), and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster, and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2 -13 differences compared to Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared to Sanger, and importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates.

Functional and multi-omics signatures of mitapivat efficacy upon activation of pyruvate kinase in red blood cells from patients with sickle cell disease.

Mitapivat, a pyruvate kinase (PK) activator, shows great potential as a sickle cell disease (SCD)- modifying therapy. Safety and efficacy of mitapivat as a long-term maintenance therapy is currently being evaluated in two open-label studies. Here we apply a comprehensive multi-omics approach to investigate the impact of activating PK on red blood cells (RBCs) from 15 SCD patients. HbSS patients were enrolled in one of the open label, extended studies (NCT04610866). Leuko-depleted RBCs obtained from fresh whole blood at baseline (visit 1, V1), prior to drug initiation and longitudinal time points over the course of the study were processed for multiomics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Mitapivat decreased 2,3-diphosphoglycerate (DPG) levels, increased adenosine triphosphate (ATP) levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S (HbS) oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of drug treatment, with minimal changes in the reticulocyte counts. The first six months of treatment also witnessed transient elevation of lysophosphatidylcholines and oxylipins with depletion in free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBCs identified benefits for glycolysis, as well as activation of the Lands cycle.

Etiologies of failed back surgery syndrome.

STUDY DESIGN: Retrospective chart review. OBJECTIVE: To report the epidemiologic data of nonsurgical and surgical etiologies of failed back surgery syndrome (FBSS) from two outpatient spine practices. SUMMARY OF BACKGROUND DATA: FBSS has been offered as a diagnosis, but this is an imprecise term encompassing a heterogeneous group of disorders that have in common pain symptoms after lumbar surgery. The current literature primarily diagnoses for the various etiologies of FBSS from a surgical perspective. To our knowledge, there is no study that investigates the myriad of surgical and nonsurgical diagnoses from a nonsurgical perspective. METHODS: Specific inclusion and exclusion criteria were developed for a list of 42 nonsurgical and surgical differential diagnoses of FBSS. The determination of which category, surgical or nonsurgical, each diagnosis was placed into depended upon the categorization of those diagnoses in previously published literature on FBSS. Each of the authors reviewed the definitions, and they came to a unanimous agreement on each diagnosis' inclusion and exclusion criteria. Data extraction was then carried out in each of the two involved institutions by using the key words discectomy, laminectomy, and fusion to identify all the patients who had any combination of low back, buttock, or lower extremity pain after lumbar discectomy surgery. These charts were then individually reviewed to extract epidemiologic data. RESULTS: A total of 267 charts were reviewed. One hundred and ninety-seven (197) charts had a complete workup. Of these, 11 (5.6%) had an unknown etiology, and 186 had a known diagnosis. Twenty-three (23) various diagnoses were identified. There was approximately an equal distribution between the incidences of nonsurgical and surgical diagnoses; 44.4% had nonsurgical diagnoses and 55.6% had surgical diagnoses. The most common diagnoses identified were spinal stenosis, internal disc disruption syndrome, recurrent/retained disc, and neural fibrosis. CONCLUSION: FBSS is a syndrome consisting of a myriad of surgical and nonsurgical etiologies. Approximately one half of FBSS patients have a surgical etiology. Approximately 95% of patients can be provided a specific diagnosis.