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The Positional Burrows–Wheeler Transform (PBWT) is a data structure that indexes haplotype sequences in a manner that enables finding maximal haplotype matches in h sequences containing w variation sites in O(hw) time. This represents a significant improvement over classical quadratic-time approaches. However, the original PBWT data structure does not allow for queries over Biobank panels that consist of several millions of haplotypes, if an index of the haplotypes must be kept entirely in memory.

In this article, we leverage the notion of r-index proposed for the BWT to present a memory-efficient method for constructing and storing the run-length encoded PBWT, and computing set maximal matches (SMEMs) queries in haplotype sequences. We implement our method, which we refer to as μ-PBWT, and evaluate it on datasets of 1000 Genome Project and UK Biobank data. Our experiments demonstrate that the μ-PBWT reduces the memory usage up to a factor of 20% compared to the best current PBWT-based indexing. In particular, μ-PBWT produces an index that stores high-coverage whole genome sequencing data of chromosome 20 in about a third of the space of its BCF file. μ-PBWT is an adaptation of techniques for the run-length compressed BWT for the PBWT (RLPBWT) and it is based on keeping in memory only a succinct representation of the RLPBWT that still allows the efficient computation of set maximal matches (SMEMs) over the original panel.

Our implementation is open source and available at https://github.com/dlcgold/muPBWT. The binary is available at https://bioconda.github.io/recipes/mupbwt/README.html.

 

Genomics analyses use large reference sequence collections, like pangenomes or taxonomic databases. SPUMONI 2 is an efficient tool for sequence classification of both short and long reads. It performs multi-class classification using a novel sampled document array. By incorporating minimizers, SPUMONI 2’s index is 65 times smaller than minimap2’s for a mock community pangenome. SPUMONI 2 achieves a speed improvement of 3-fold compared to SPUMONI and 15-fold compared to minimap2. We show SPUMONI 2 achieves an advantageous mix of accuracy and efficiency in practical scenarios such as adaptive sampling, contamination detection and multi-class metagenomics classification.

 

Characterization of antibiotic resistance genes (ARGs) from high-throughput sequencing data of metagenomics and cultured bacterial samples is a challenging task, with the need to account for both computational (e.g., string algorithms) and biological (e.g., gene transfers, rearrangements) aspects. Curated ARG databases exist together with assorted ARG classification approaches (e.g., database alignment, machine learning). Besides ARGs that naturally occur in bacterial strains or are acquired through mobile elements, there are chromosomal genes that can render a bacterium resistant to antibiotics through point mutations, i.e., ARG variants (ARGVs). While ARG repositories also collect ARGVs, there are only a few tools that are able to identify ARGVs from metagenomics and high throughput sequencing data, with a number of limitations (e.g., pre-assembly,a posterioriverification of mutations, or specification of species). In this work we present thek-mer, i.e., strings of fixed lengthk, ARGV analyzer – KARGVA – an open-source, multi-platform tool that provides: (i) anad hoc, large ARGV database derived from multiple sources; (ii) input capability for various types of high-throughput sequencing data; (iii) a three-way, hash-based,k-mer search setup to process data efficiently, linkingk-mers to ARGVs,k-mers to point mutations, and ARGVs tok-mers, respectively; (iv) a statistical filter on sequence classification to reduce type I and II errors. On semi-synthetic data, KARGVA provides very high accuracy even in presence of high sequencing errors or mutations (99.2 and 86.6% accuracy within 1 and 5% base change rates, respectively), and genome rearrangements (98.2% accuracy), with robust performance onad hocfalse positive sets. On data from the worldwide MetaSUB consortium, comprising 3,700+ metagenomics experiments, KARGVA identifies more ARGVs than Resistance Gene Identifier (4.8x) and PointFinder (6.8x), yet all predictions are below the expected false positive estimates. The prevalence of ARGVs is correlated to ARGs but ecological characteristics do not explain well ARGV variance. KARGVA is publicly available athttps://github.com/DataIntellSystLab/KARGVAunder MIT license.

 

Nanopore technology enables portable, real-time sequencing of microbial populations from clinical and ecological samples. An emerging healthcare application for Nanopore includes point-of-care, timely identification of antibiotic resistance genes (ARGs) to help developing targeted treatments of bacterial infections, and monitoring resistant outbreaks in the environment. While several computational tools exist for classifying ARGs from sequencing data, to date (2022) none have been developed for mobile devices. We present here KARGAMobile, a mobile app for portable, real-time, easily interpretable analysis of ARGs from Nanopore sequencing. KARGAMobile is the porting of an existing ARG identification tool named KARGA; it retains the same algorithmic structure, but it is optimized for mobile devices. Specifically, KARGAMobile employs a compressed ARG reference database and different internal data structures to save RAM usage. The KARGAMobile app features a friendly graphical user interface that guides through file browsing, loading, parameter setup, and process execution. More importantly, the output files are post-processed to create visual, printable and shareable reports, aiding users to interpret the ARG findings. The difference in classification performance between KARGAMobile and KARGA is minimal (96.2% vs . 96.9% f-measure on semi-synthetic datasets of 1 million reads with known resistance ground truth). Using real Nanopore experiments, KARGAMobile processes on average 1 GB data every 23–48 min (targeted sequencing - metagenomics), with peak RAM usage below 500MB, independently from input file sizes, and an average temperature of 49°C after 1 h of continuous data processing. KARGAMobile is written in Java and is available at https://github.com/Ruiz-HCI-Lab/KargaMobile under the MIT license. 

Abstract Biological and biomedical research is increasingly conducted in large, interdisciplinary collaborations to address problems with significant societal impact, such as reducing antibiotic resistance, identifying disease sub-types, and identifying genes that control for drought tolerance in plants. Many of these projects are data driven and involve the collection and analysis of biological data at a large-scale. As a result, life-science projects, which are frequently diverse, large and geographically dispersed, have created unique challenges for collaboration and training. We examine the communication and collaboration challenges in multidisciplinary research through an interview study with 20 life-science researchers. Our results show that both the inclusion of multiple disciplines and differences in work culture influence collaboration in life science. Using these results, we discuss opportunities and implications for designing solutions to better support collaborative tasks and workflows of life scientists. In particular, we show that life science research is increasingly conducted in large, multi-institutional collaborations, and these large groups rely on “mutual respect” and collaboration. However, we found that the interdisciplinary nature of these projects cause technical language barriers and differences in methodology affect trust. We use these findings to guide our recommendations for technology to support life science. We also present recommendations for life science research training programs and note the necessity for incorporating training in project management, multiple language, and discipline culture. 

Abstract Background Metagenomic data can be used to profile high-importance genes within microbiomes. However, current metagenomic workflows produce data that suffer from low sensitivity and an inability to accurately reconstruct partial or full genomes, particularly those in low abundance. These limitations preclude colocalization analysis, i.e., characterizing the genomic context of genes and functions within a metagenomic sample. Genomic context is especially crucial for functions associated with horizontal gene transfer (HGT) via mobile genetic elements (MGEs), for example antimicrobial resistance (AMR). To overcome this current limitation of metagenomics, we present a method for comprehensive and accurate reconstruction of antimicrobial resistance genes (ARGs) and MGEs from metagenomic DNA, termed t arget- e nriched l ong-read seq uencing (TELSeq). Results Using technical replicates of diverse sample types, we compared TELSeq performance to that of non-enriched PacBio and short-read Illumina sequencing. TELSeq achieved much higher ARG recovery (>1,000-fold) and sensitivity than the other methods across diverse metagenomes, revealing an extensive resistome profile comprising many low-abundance ARGs, including some with public health importance. Using the long reads generated by TELSeq, we identified numerous MGEs and cargo genes flanking the low-abundance ARGs, indicating that these ARGs could be transferred across bacterial taxa via HGT. Conclusions TELSeq can provide a nuanced view of the genomic context of microbial resistomes and thus has wide-ranging applications in public, animal, and human health, as well as environmental surveillance and monitoring of AMR. Thus, this technique represents a fundamental advancement for microbiome research and application. 

Abstract Antimicrobial resistance (AMR) is considered a critical threat to public health, and genomic/metagenomic investigations featuring high-throughput analysis of sequence data are increasingly common and important. We previously introduced MEGARes, a comprehensive AMR database with an acyclic hierarchical annotation structure that facilitates high-throughput computational analysis, as well as AMR++, a customized bioinformatic pipeline specifically designed to use MEGARes in high-throughput analysis for characterizing AMR genes (ARGs) in metagenomic sequence data. Here, we present MEGARes v3.0, a comprehensive database of published ARG sequences for antimicrobial drugs, biocides, and metals, and AMR++ v3.0, an update to our customized bioinformatic pipeline for high-throughput analysis of metagenomic data (available at MEGLab.org). Database annotations have been expanded to include information regarding specific genomic locations for single-nucleotide polymorphisms (SNPs) and insertions and/or deletions (indels) when required by specific ARGs for resistance expression, and the updated AMR++ pipeline uses this information to check for presence of resistance-conferring genetic variants in metagenomic sequenced reads. This new information encompasses 337 ARGs, whose resistance-conferring variants could not previously be confirmed in such a manner. In MEGARes 3.0, the nodes of the acyclic hierarchical ontology include 4 antimicrobial compound types, 59 resistance classes, 233 mechanisms and 1448 gene groups that classify the 8733 accessions.