Computational Tools for the Analysis of Uncultivated Phage Genomes.

Over a century of bacteriophage research has uncovered a plethora of fundamental aspects of their biology, ecology, and evolution. Furthermore, the introduction of community-level studies through metagenomics has revealed unprecedented insights on the impact that phages have on a range of ecological and physiological processes. It was not until the introduction of viral metagenomics that we began to grasp the astonishing breadth of genetic diversity encompassed by phage genomes. Novel phage genomes have been reported from a diverse range of biomes at an increasing rate, which has prompted the development of computational tools that support the multilevel characterization of these novel phages based solely on their genome sequences. The impact of these technologies has been so large that, together with MAGs (Metagenomic Assembled Genomes), we now have UViGs (Uncultivated Viral Genomes), which are now officially recognized by the International Committee for the Taxonomy of Viruses (ICTV), and new taxonomic groups can now be created based exclusively on genomic sequence information. Even though the available tools have immensely contributed to our knowledge of phage diversity and ecology, the ongoing surge in software programs makes it challenging to keep up with them and the purpose each one is designed for. Therefore, in this review, we describe a comprehensive set of currently available computational tools designed for the characterization of phage genome sequences, focusing on five specific analyses: (i) assembly and identification of phage and prophage sequences, (ii) phage genome annotation, (iii) phage taxonomic classification, (iv) phage-host interaction analysis, and (v) phage microdiversity.

Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments.

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetics method in plant functional genomics and breeding to identify mutagenized individuals with improved behavior for a trait of interest. Pooled high throughput sequencing (HTS) of the targeted genes allows efficient identification and sample assignment of variants within genes of interest in hundreds of individuals. Although TILLING has been used successfully in different crops and even applied to natural populations, one of the main issues for a successful TILLING experiment is that most currently available bioinformatics tools for variant detection are not designed to identify mutations with low frequencies in pooled samples or to perform sample identification from variants identified in overlapping pools. Our research group maintains the Next Generation Sequencing Experience Platform (NGSEP), an open source solution for analysis of HTS data. In this manuscript, we present three novel components within NGSEP to facilitate the design and analysis of TILLING experiments: a pooled variants detector, a sample identifier from variants detected in overlapping pools and a simulator of TILLING experiments. A new implementation of the NGSEP calling model for variant detection allows accurate detection of low frequency mutations within pools. The samples identifier implements the process to triangulate the mutations called within overlapping pools in order to assign mutations to single individuals whenever possible. Finally, we developed a complete simulator of TILLING experiments to enable benchmarking of different tools and to facilitate the design of experimental alternatives varying the number of pools and individuals per pool. Simulation experiments based on genes from the common bean genome indicate that NGSEP provides similar accuracy and better efficiency than other tools to perform pooled variants detection. To the best of our knowledge, NGSEP is currently the only tool that generates individual assignments of the mutations discovered from the pooled data. We expect that this development will be of great use for different groups implementing TILLING as an alternative for plant breeding and even to research groups performing pooled sequencing for other applications.