Early Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Variants Using Traveler-based Genomic Surveillance at 4 US Airports, September 2021-January 2022.

We enrolled arriving international air travelers in a severe acute respiratory syndrome coronavirus 2 genomic surveillance program. We used molecular testing of pooled nasal swabs and sequenced positive samples for sublineage. Traveler-based surveillance provided early-warning variant detection, reporting the first US Omicron BA.

Variant abundance estimation for SARS-CoV-2 in wastewater using RNA-Seq quantification.

Effectively monitoring the spread of SARS-CoV-2 variants is essential to efforts to counter the ongoing pandemic. Wastewater monitoring of SARS-CoV-2 RNA has proven an effective and efficient technique to approximate COVID-19 case rates in the population. Predicting variant abundances from wastewater, however, is technically challenging.

Designing and Implementing NGS Tests for Inherited Disorders: A Practical Framework with Step-by-Step Guidance for Clinical Laboratories.

Comprehensive next-generation sequencing (NGS) tests are increasingly used as first-line tests in the evaluation of patients with suspected heritable disease. Despite major technical simplifications, these assays still pose significant challenges for molecular testing laboratories. Existing professional guidelines and recommendations provide a framework for laboratories implementing such tests, but in-depth, concrete guidance is generally not provided.

A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.

Background: Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance.

Methods: A multicenter study was conducted to validate an updated assay design for 454 Life Sciences' GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity.

Validation of a next-generation-sequencing cancer panel for use in the clinical laboratory.

Context: Next-generation sequencing allows for high-throughput processing and sensitive variant detection in multiple genes from small samples. For many diseases, including cancer, a comprehensive mutational profile of a targeted list of genes can be used to simultaneously inform patient care, establish eligibility for ongoing clinical trials, and further research.

Objective: To validate a pan-cancer, next-generation-sequencing assay for use in the clinical laboratory.

Low-frequency NNRTI-resistant HIV-1 variants and relationship to mutational load in antiretroviral-naïve subjects.

Low-frequency HIV variants possessing resistance mutations against non‑nucleoside reverse transcriptase inhibitors (NNRTI), especially at HIV reverse transcriptase (RT) amino acid (aa) positions K103 and Y181, have been shown to adversely affect treatment response. Therapeutic failure correlates with both the mutant viral variant frequency and the mutational load. We determined the prevalence of NNRTI resistance mutations at several RT aa positions in viruses from 204 antiretroviral (ARV)-naïve HIV-infected individuals using deep sequencing, and examined the relationship between mutant variant frequency and mutational load for those variants.

Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements.

B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition.

An international multicenter study on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing.

The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study.

High-resolution antibody dynamics of vaccine-induced immune responses.

The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers.

The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies.

Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134.

Convergent antibody signatures in human dengue.

Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection.

Next generation sequencing reveals the association of DRB3*02:02 with type 1 diabetes.

The primary associations of the HLA class II genes, HLA-DRB1 and HLA-DQB1, and the class I genes, HLA-A and HLA-B, with type 1 diabetes (T1D) are well established. However, the role of polymorphism at the HLA-DRB3, HLA-DRB4, and HLA-DRB5 loci remains unclear. In two separate studies, one of 500 subjects and 500 control subjects and one of 366 DRB1*03:01-positive samples from selected multiplex T1D families, we used Roche 454 sequencing with Conexio Genomics ASSIGN ATF 454 HLA genotyping software analysis to analyze sequence variation at these three HLA-DRB loci.

Therapy failure resulting from superinfection by a drug-resistant HIV variant.

Background: HIV-1-infected patients can be superinfected with additional HIV-1 variants. Therapy failure can be the consequence of an infection with a resistant strain.

Methods: A patient was diagnosed with a recent HIV-1 infection in April 2005 and subsequently clinically monitored.

Virologic failures on initial boosted-PI regimen infrequently possess low-level variants with major PI resistance mutations by ultra-deep sequencing.

Background: It is unknown whether HIV-positive patients experiencing virologic failure (VF) on boosted-PI (PI/r) regimens without drug resistant mutations (DRM) by standard genotyping harbor low-level PI resistant variants. CASTLE compared the efficacy of atazanavir/ritonavir (ATV/r) with lopinavir/ritonavir (LPV/r), each in combination with TVD in ARV-naïve subjects.

Objective: To determine if VF on an initial PI/r-based regimen possess low-level resistant variants that may affect a subsequent PI-containing regimen.

Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated.

The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies.

High-throughput sequencing of PCR products tagged with universal primers using 454 life sciences systems.

This unit describes a method to convert PCR products (amplicons) flanked by universal M13 primers into a library for use on all 454 Sequencing Systems (454 Life Sciences, a Roche Company). This is especially useful for simultaneous sequencing and analysis of large numbers of amplicons or for the detection of minor variations within the amplified products. The method described here involves preparing a library of DNA with specific primers containing adaptor sequences recognized by the GS Junior System sequencing process.

Use of dried-blood-spot samples and in-house assays to identify antiretroviral drug resistance in HIV-infected children in resource-constrained settings.

Monitoring HIV drug resistance is an important component of the World Health Organization's global HIV program. HIV drug resistance testing is optimal with commercially available clinically validated test kits using plasma; however, that type of testing may not be feasible or affordable in resource-constrained settings. HIV genotyping from dried blood spots (DBS) with noncommercial (in-house) assays may facilitate the capture of HIV drug resistance outcomes in resource-constrained settings but has had varying rates of success.

Prevalence of low-level HIV-1 variants with reverse transcriptase mutation K65R and the effect of antiretroviral drug exposure on variant levels.

Background: It has been reported that treatment-naive individuals infected with HIV-1 subtype C may be more likely to harbour viral variants possessing a K65R reverse transcriptase gene mutation. The objectives of this study were to determine the prevalence of low-level K65R variants within different HIV-1 subtypes and to assess the effects of antiretroviral exposure on K65R variant levels.

Methods: Treatment-naive individuals infected with different HIV-1 subtypes were genotyped by ultra-deep sequencing.

Prevalence and clinical significance of HIV drug resistance mutations by ultra-deep sequencing in antiretroviral-naïve subjects in the CASTLE study.

Background: CASTLE compared the efficacy of atazanavir/ritonavir with lopinavir/ritonavir, each in combination with tenofovir-emtricitabine in ARV-naïve subjects from 5 continents.

Objectives: Determine the baseline rate and clinical significance of TDR mutations using ultra-deep sequencing (UDS) in ARV-naïve subjects in CASTLE.

Methods: A case control study was performed on baseline samples for all 53 subjects with virologic failures (VF) at Week 48 and 95 subjects with virologic successes (VS) randomly selected and matched by CD4 count and viral load.