# The OBITools

The OBITools4 are programs specifically designed for analyzing NGS data in a DNA metabarcoding context, taking into account taxonomic information. It is distributed as an open source software available on the following website: http://metabarcoding.org/obitools4.

## Aims of OBITools

DNA metabarcoding is an efficient approach for biodiversity studies (Taberlet et al. 2012). Originally mainly developed by microbiologists (e.g. Sogin et al. 2006), it is now widely used for plants (e.g. Sønstebø et al. 2010; Yoccoz et al. 2012; Parducci et al. 2012) and animals from meiofauna (e.g. Chariton et al. 2010; Baldwin et al. 2013) to larger organisms (e.g. Andersen et al. 2012; Thomsen et al. 2012). Interestingly, this method is not limited to sensu stricto biodiversity surveys, but it can also be implemented in other ecological contexts such as for herbivore (e.g. Valentini et al. 2009; Kowalczyk et al. 2011) or carnivore (e.g. Deagle, Kirkwood, and Jarman 2009; Shehzad et al. 2012) diet analyses.

Whatever the biological question under consideration, the DNA metabarcoding methodology relies heavily on next-generation sequencing (NGS), and generates considerable numbers of DNA sequence reads (typically million of reads). Manipulation of such large datasets requires dedicated programs usually running on a Unix system. Unix is an operating system, whose first version was created during the sixties. Since its early stages, it is dedicated to scientific computing and includes a large set of simple tools to efficiently process text files. Most of those programs can be viewed as filters extracting information from a text file to create a new text file. These programs process text files as streams, line per line, therefore allowing computation on a huge dataset without requiring a large memory. Unix programs usually print their results to their standard output (stdout), which by default is the terminal, so the results can be examined on screen. The main philosophy of the Unix environment is to allow easy redirection of the stdout either to a file, for saving the results, or to the standard input (stdin) of a second program thus allowing to easily create complex processing from simple base commands. Access to Unix computers is increasingly easier for scientists nowadays. Indeed, the Linux operating system, an open source version of Unix, can be freely installed on every PC machine and the MacOS operating system, running on Apple computers, is also a Unix system. The OBITools programs imitate Unix standard programs because they usually act as filters, reading their data from text files or the stdin and writing their results to the stdout. The main difference with classical Unix programs is that text files are not analyzed line per line but sequence record per sequence record (see below for a detailed description of a sequence record). Compared to packages for similar purposes like mothur (Schloss et al. 2009) or QIIME (Caporaso et al. 2010), the OBITools mainly rely on filtering and sorting algorithms. This allows users to set up versatile data analysis pipelines (Figure 1), adjustable to the broad range of DNA metabarcoding applications. The innovation of the OBITools is their ability to take into account the taxonomic annotations, ultimately allowing sorting and filtering of sequence records based on the taxonomy.

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