Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies.

SUMMARY: Retroviruses and their vector derivatives integrate semi-randomly in the genome of host cells and are inherited by their progeny as stable genetic marks. The retrieval and mapping of the sequences flanking the virus-host DNA junctions allows the identification of insertion sites in gene therapy or virally infected patients, essential for monitoring the evolution of genetically modified cells in vivo. However, since ∼30% of insertions land in low complexity or repetitive regions of the host cell genome, they cannot be correctly assigned and are currently discarded, limiting the accuracy and predictive power of clonal tracking studies. Here, we present γ-TRIS, a new graph-based genome-free alignment tool for identifying insertion sites even if embedded in low complexity regions. By using γ-TRIS to reanalyze clinical studies, we observed improvements in clonal quantification and tracking. AVAILABILITY AND IMPLEMENTATION: Source code at https://bitbucket.org/bereste/g-tris. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: RNA-Seq is an increasing used methodology to study either coding and non-coding RNA expression. There are many software tools available for each phase of the RNA-Seq analysis and each of them uses different algorithms. Furthermore, the analysis consists of several steps regarding alignment (primary-analysis), quantification, differential analysis (secondary-analysis) and any tertiary-analysis and can therefore be time-consuming to deal with each step separately, in addition to requiring a computer knowledge. For this reason, the development of an automated pipeline that allows the entire analysis to be managed through a single initial command and that is easy to use even for those without computer skills can be useful. Faced with the vast availability of RNA-Seq analysis tools, it is first of all necessary to select a limited number of pipelines to include. For this purpose, we compared eight pipelines obtained by combining the most used tools and for each one we evaluated peak of RAM, time, sensitivity and specificity. RESULTS: The pipeline with shorter times, lower consumption of RAM and higher sensitivity is the one consisting in HISAT2 for alignment, featureCounts for quantification and edgeR for differential analysis. Here, we developed ARPIR, an automated pipeline that recurs by default to the cited pipeline, but it also allows to choose, between different tools, those of the pipelines having the best performances. CONCLUSIONS: ARPIR allows the analysis of RNA-Seq data from groups undergoing different treatment allowing multiple comparisons in a single launch and can be used either for paired-end or single-end analysis. All the required prerequisites can be installed via a configuration script and the analysis can be launched via a graphical interface or by a template script. In addition, ARPIR makes a final tertiary-analysis that includes a Gene Ontology and Pathway analysis. The results can be viewed in an interactive Shiny App and exported in a report (pdf, word or html formats). ARPIR is an efficient and easy-to-use tool for RNA-Seq analysis from quality control to Pathway analysis that allows you to choose between different pipelines.

BACKGROUND: Bioinformatics tools designed to identify lentiviral or retroviral vector insertion sites in the genome of host cells are used to address the safety and long-term efficacy of hematopoietic stem cell gene therapy applications and to study the clonal dynamics of hematopoietic reconstitution. The increasing number of gene therapy clinical trials combined with the increasing amount of Next Generation Sequencing data, aimed at identifying integration sites, require both highly accurate and efficient computational software able to correctly process "big data" in a reasonable computational time. RESULTS: Here we present VISPA2 (Vector Integration Site Parallel Analysis, version 2), the latest optimized computational pipeline for integration site identification and analysis with the following features: (1) the sequence analysis for the integration site processing is fully compliant with paired-end reads and includes a sequence quality filter before and after the alignment on the target genome; (2) an heuristic algorithm to reduce false positive integration sites at nucleotide level to reduce the impact of Polymerase Chain Reaction or trimming/alignment artifacts; (3) a classification and annotation module for integration sites; (4) a user friendly web interface as researcher front-end to perform integration site analyses without computational skills; (5) the time speedup of all steps through parallelization (Hadoop free). CONCLUSIONS: We tested VISPA2 performances using simulated and real datasets of lentiviral vector integration sites, previously obtained from patients enrolled in a hematopoietic stem cell gene therapy clinical trial and compared the results with other preexisting tools for integration site analysis. On the computational side, VISPA2 showed a > 6-fold speedup and improved precision and recall metrics (1 and 0.97 respectively) compared to previously developed computational pipelines. These performances indicate that VISPA2 is a fast, reliable and user-friendly tool for integration site analysis, which allows gene therapy integration data to be handled in a cost and time effective fashion. Moreover, the web access of VISPA2 ( http://openserver.itb.cnr.it/vispa/ ) ensures accessibility and ease of usage to researches of a complex analytical tool. We released the source code of VISPA2 in a public repository ( https://bitbucket.org/andreacalabria/vispa2 ).

BACKGROUND: Many biological laboratories that deal with genomic samples are facing the problem of sample tracking, both for pure laboratory management and for efficiency. Our laboratory exploits PCR techniques and Next Generation Sequencing (NGS) methods to perform high-throughput integration site monitoring in different clinical trials and scientific projects. Because of the huge amount of samples that we process every year, which result in hundreds of millions of sequencing reads, we need to standardize data management and tracking systems, building up a scalable and flexible structure with web-based interfaces, which are usually called Laboratory Information Management System (LIMS). METHODS: We started collecting end-users' requirements, composed of desired functionalities of the system and Graphical User Interfaces (GUI), and then we evaluated available tools that could address our requirements, spanning from pure LIMS to Content Management Systems (CMS) up to enterprise information systems. Our analysis identified ADempiere ERP, an open source Enterprise Resource Planning written in Java J2EE, as the best software that also natively implements some highly desirable technological advances, such as the high usability and modularity that grants high use-case flexibility and software scalability for custom solutions. RESULTS: We extended and customized ADempiere ERP to fulfil LIMS requirements and we developed adLIMS. It has been validated by our end-users verifying functionalities and GUIs through test cases for PCRs samples and pre-sequencing data and it is currently in use in our laboratories. adLIMS implements authorization and authentication policies, allowing multiple users management and roles definition that enables specific permissions, operations and data views to each user. For example, adLIMS allows creating sample sheets from stored data using available exporting operations. This simplicity and process standardization may avoid manual errors and information backtracking, features that are not granted using track recording on files or spreadsheets. CONCLUSIONS: adLIMS aims to combine sample tracking and data reporting features with higher accessibility and usability of GUIs, thus allowing time to be saved on doing repetitive laboratory tasks, and reducing errors with respect to manual data collection methods. Moreover, adLIMS implements automated data entry, exploiting sample data multiplexing and parallel/transactional processing. adLIMS is natively extensible to cope with laboratory automation through platform-dependent API interfaces, and could be extended to genomic facilities due to the ERP functionalities.