MOTIVATION: Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) has emerged as a central approach for studying T cell and B cell receptor populations, and is now an important component of studies of autoimmunity, immune responses to pathogens, vaccines, allergens, and cancers, and for antibody discovery. When amplifying the rearranged V(D)J genes encoding antigen receptors, each cycle of the Polymerase Chain Reaction (PCR) can produce spurious "chimeric" hybrids of two or more different template sequences. While the generation of chimeras is well understood in bacterial and viral sequencing, and there are dedicated tools to detect such sequences in bacterial and viral datasets, this is not the case for AIRR-seq. Further, the process that results in immune receptor sequences has domain-specific challenges, such as somatic hypermutation (SHM), and domain-specific opportunities, such as relatively well-known germline gene "reference" sequences. RESULTS: Here, we describe CHMMAIRRa, a hidden Markov model for detecting chimeric sequences in AIRR-seq data, that specifically models SHM and incorporates germline reference sequences. We use simulations to characterize the performance of CHMMAIRRa and compare it to existing methods from other domains, we test the effect of PCR conditions on chimerism using IgM libraries generated in this study, and we apply CHMMAIRRa to four published AIRR-seq datasets to show the extent and impact of artifactual chimerism. AVAILABILITY AND IMPLEMENTATION: CHMMAIRRa is published on the Julia package registry and is available at https://github.com/MurrellGroup/CHMMAIRRa.jl (DOI: 10.5281/zenodo.17279881). The core HMM implementation is available at https://github.com/MurrellGroup/CHMMera.jl (DOI: 10.5281/zenodo.17279998), and the scripts used to generate the results in this paper at https://github.com/MurrellGroup/CHMMAIRRaAnalyses (DOI: 10.5281/zenodo.17281446).

Motivation: Protein coding genes can be studied using long-read next generation sequencing. However, high rates of indel sequencing errors are problematic, corrupting the reading frame. Even the consensus of multiple independent sequence reads retains indel errors. To solve this problem, we introduce Reference-Informed Frame-Resolving multiple-Alignment Free template inference algorithm (RIFRAF), a sequence consensus algorithm that takes a set of error-prone reads and a reference sequence and infers an accurate in-frame consensus. RIFRAF uses a novel structure, analogous to a two-layer hidden Markov model: the consensus is optimized to maximize alignment scores with both the set of noisy reads and with a reference. The template-to-reads component of the model encodes the preponderance of indels, and is sensitive to the per-base quality scores, giving greater weight to more accurate bases. The reference-to-template component of the model penalizes frame-destroying indels. A local search algorithm proceeds in stages to find the best consensus sequence for both objectives. Results: Using Pacific Biosciences SMRT sequences from an HIV-1 env clone, NL4-3, we compare our approach to other consensus and frame correction methods. RIFRAF consistently finds a consensus sequence that is more accurate and in-frame, especially with small numbers of reads. It was able to perfectly reconstruct over 80% of consensus sequences from as few as three reads, whereas the best alternative required twice as many. RIFRAF is able to achieve these results and keep the consensus in-frame even with a distantly related reference sequence. Moreover, unlike other frame correction methods, RIFRAF can detect and keep true indels while removing erroneous ones. Availability and implementation: RIFRAF is implemented in Julia, and source code is publicly available at https://github.com/MurrellGroup/Rifraf.jl. Supplementary information: Supplementary data are available at Bioinformatics online.

Next generation sequencing of viral populations has advanced our understanding of viral population dynamics, the development of drug resistance, and escape from host immune responses. Many applications require complete gene sequences, which can be impossible to reconstruct from short reads. HIV env, the protein of interest for HIV vaccine studies, is exceptionally challenging for long-read sequencing and analysis due to its length, high substitution rate, and extensive indel variation. While long-read sequencing is attractive in this setting, the analysis of such data is not well handled by existing methods. To address this, we introduce FLEA (Full-Length Envelope Analyzer), which performs end-to-end analysis and visualization of long-read sequencing data. FLEA consists of both a pipeline (optionally run on a high-performance cluster), and a client-side web application that provides interactive results. The pipeline transforms FASTQ reads into high-quality consensus sequences (HQCSs) and uses them to build a codon-aware multiple sequence alignment. The resulting alignment is then used to infer phylogenies, selection pressure, and evolutionary dynamics. The web application provides publication-quality plots and interactive visualizations, including an annotated viral alignment browser, time series plots of evolutionary dynamics, visualizations of gene-wide selective pressures (such as dN/dS) across time and across protein structure, and a phylogenetic tree browser. We demonstrate how FLEA may be used to process Pacific Biosciences HIV env data and describe recent examples of its use. Simulations show how FLEA dramatically reduces the error rate of this sequencing platform, providing an accurate portrait of complex and variable HIV env populations. A public instance of FLEA is hosted at http://flea.datamonkey.org. The Python source code for the FLEA pipeline can be found at https://github.com/veg/flea-pipeline. The client-side application is available at https://github.com/veg/flea-web-app. A live demo of the P018 results can be found at http://flea.murrell.group/view/P018.

Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals--a cure and a vaccine--remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes) for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab), determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license), documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.

The evolution of substitutions conferring drug resistance to HIV-1 is both episodic, occurring when patients are on antiretroviral therapy, and strongly directional, with site-specific resistant residues increasing in frequency over time. While methods exist to detect episodic diversifying selection and continuous directional selection, no evolutionary model combining these two properties has been proposed. We present two models of episodic directional selection (MEDS and EDEPS) which allow the a priori specification of lineages expected to have undergone directional selection. The models infer the sites and target residues that were likely subject to directional selection, using either codon or protein sequences. Compared to its null model of episodic diversifying selection, MEDS provides a superior fit to most sites known to be involved in drug resistance, and neither one test for episodic diversifying selection nor another for constant directional selection are able to detect as many true positives as MEDS and EDEPS while maintaining acceptable levels of false positives. This suggests that episodic directional selection is a better description of the process driving the evolution of drug resistance.