Journal:A polyglot approach to bioinformatics data integration: A phylogenetic analysis of HIV-1

Abstract

As sequencing technologies continue to drop in price and increase in throughput, new challenges emerge for the management and accessibility of genomic sequence data. We have developed a pipeline for facilitating the storage, retrieval, and subsequent analysis of molecular data, integrating both sequence and metadata. Taking a polyglot approach involving multiple languages, libraries, and persistence mechanisms, sequence data can be aggregated from publicly available and local repositories. Data are exposed in the form of a RESTful web service, formatted for easy querying, and retrieved for downstream analyses. As a proof of concept, we have developed a resource for annotated HIV-1 sequences. Phylogenetic analyses were conducted for >6,000 HIV-1 sequences revealing spatial and temporal factors influence the evolution of the individual genes uniquely. Nevertheless, signatures of origin can be extrapolated even despite increased globalization. The approach developed here can easily be customized for any species of interest.

Keywords: polyglot programming, RESTful web service, phylogenetics

Introduction

The increased throughput, coupled with reduced cost and time, of contemporary sequencing technologies has led to a surge in the number of publicly available, complete, annotated genomic sequences. For smaller viral species, it is now feasible to not only produce a single genome for a species but also capture the diversity present in an ecological niche, the focus of numerous metagenomic studies as well as more targeted investigations. Furthermore, next-generation sequencing technologies have tremendous potential for the future of diagnostics and subsequent treatment choices, particularly for viral infections. The sensitivity of deep sequencing can capture even rare variants in mixed infections as well as quasispecies. Investigation of the viable variations within a viral species not only provides insight into the evolutionary history of a species but also unveils putative avenues for targeted therapies, such as small interfering RNAs and control strategies.

Molecular biology is now plagued with the challenges facing numerous other fields – big data. Cloud-based solutions, e.g. CloudBurst, Atlas2, and Rainbow, have provided much needed leverage to meet these demands, facilitating large-scale sequence analyses, while also introducing new difficulties. Furthermore, noSQL databases afford a streamlined solution to both manage large datasets and simplify data retrieval and subsequent analysis. The added benefit of agility and scalability of noSQL databases is ideal for the rapidly advancing trends in DNA sequencing technologies, and it is thus not surprising that noSQL databases have been gaining traction in molecular studies.

With the increase in the amount of publicly available genomic sequence data, progress can be stymied by the simple task of collecting sequence data and associated, relevant metadata. In an effort to facilitate the aggregation and management of genomic data for subsequent analyses, we have developed a polyglot approach involving multiple languages (Python and Scala), libraries (Flask and BioJavaX), and persistence mechanisms (text files and MongoDB NoSQL databases). Individual genes or all genes for a given species can be examined beyond just the sequence itself, including information regarding, for instance, the location and date of isolation.

The code developed is agile; it can be applied for any organism of interest to the user. The approach can be customized for any species of interest. The presented solution is developed with downstream evolutionary analyses in mind, as shown by a proof-of-concept study of the evolution of HIV. Our investigation into the three main HIV genes: gag, pol, and env, reveals spatial and temporal factors influence the evolution of the individual genes uniquely. The web service for the HIV collection (as well as other datasets investigated by the authors) is publicly available at http://hivdb.cs.luc.edu, and the scripts for generating such a data collection are publicly available at https://github.com/LoyolaChicagoCode/hiv-biojava-scala.

Results and discussion

Data pipeline for collecting sequence data

Code has been developed to aggregate genomic sequence data and available sequence metadata for subsequent analyses. Figure 1 summarizes this process. All complete and partial genome sequences were parsed and separated into individual folders for each gene via a Scala parser utilizing the BioJavaX library. Each sequence was stored in its gene folder with any relevant metadata available within the GenBank file. The generated folders for each of the parsed genes were then pipelined through several python scripts in order to accomplish several post-processing tasks. First, duplicate gene sequences parsed from the same genome were removed. Second, the gene folders were used to create FASTA-formatted records for each of the gene sequences with any necessary metadata stored in the resulting record’s FASTA header. Finally, the PyMongo library was used to insert each of the final FASTA records within our publicly exposed MongoDB database.

Figure 1. Schematic of data pipeline and access

Genomic sequence data can then be accessed via a RESTful web service located at http://hivdb.cs.luc.edu. This architecture allows our service to be easily and efficiently accessed by any future data consumers via common web protocols. Data can be queried based upon attributes regarding the source of the data. For example, as shown in Figure 2, the gag gene sequences from strains isolated in the USA can be accessed via the web service. The user can specify search criteria including a year (or range of years) of isolation, the location of isolation, and/or accession number. Sequences meeting the user’s search criteria can be returned to the web via the Query button or downloaded. All sequence results are in FASTA format for subsequent analysis, such as sequence alignment, primer development, and phylogenetic analysis.

Figure 2. Gene sequence data presented through RESTful web service. Users can query for specific information, eg, as shown here are HIV isolates from the USA, via the Query button or download sequence files in FASTA format meeting their search criteria.

The pipeline has been developed to facilitate users to create repositories for an organism(s) of interest as well as queryable aspects of the sequence annotations. Users need only supply sequences and select attributes and/or genes of interest (otherwise all attributes and genes will be selected). Data are automatically processed. Furthermore, the pipeline is not restricted to publicly available data; any GenBank-formatted file (public or private) can be included. Given the increased throughput of contemporary sequencing technologies and the decreased cost in sequencing runs, whole genome sequencing is being conducted at unprecedented rates. As such, researchers sequencing novel strains or isolates can incorporate their strains into the data repository once GenBank files are generated. Although this pipeline has been employed by the authors for the analysis of several different taxa, the RESTful web service presented here includes publicly available data for HIV-1 sequences.

Case study: Investigation of the evolution of HIV-1

All publicly available complete and near-complete HIV-1 genomic sequences, totaling more than 6,000 sequences, were retrieved from the National Center for Biotechnology Information (NCBI) and processed by our pipeline (see the "Methods and materials" section). Individual gene sequences are publicly available at http://hivdb.cs.luc.edu. Data are accessible in FASTA format to facilitate downstream analyses. To incorporate the metadata collected, including country and date of isolation, this information has been integrated into the FASTA record header. HIV-1 sequences were selected as a proof of concept for this tool as HIV sequences are among the most well curated, thanks in large part to efforts such as those at the Los Alamos National Laboratory’s HIV sequence database.

Previously, phylogenetics has shed light on the origin of HIV and played a key role in identifying recombination events. As previous molecular studies have shown, the evolutionary history of the HIV-1 lineage includes three groups (M, N, and O) representative of separate transfers from chimpanzees. Focusing on the three HIV genes gag, pol, and env, the hivdb data repository was queried for coding regions isolated from the same country as well as globally over a particular time period. Host, immunological and antiretroviral drug selection pressures have shaped much of the diversity observed within these three genes. For instance, the investigation of the HIV gag gene sequences from the USA (2,048 sequences: 1990–2011) and Thailand (872 sequences: 2000–2011) is shown in Figure 3A and B, respectively. The phylogenic trees derived for different geographic regions revealed different tree topologies as expected. Sequences isolated during 2005 in the USA exhibit significant sequence variation, including a number of sequences which are distinctly different from sequences isolated during any other year (Fig. 3A). These two gag trees reveal a general trend observed for other countries and other genes: sequences isolated during the same year do not necessarily group together or exhibit a ladder-like topology frequently observed for intra-host HIV phylogenies; this is in concordance with previous HIV survey findings that multiple lineages coexist at any given time.

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added.