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According to current estimations, 1.5 to 5.1 million species belong to the Fungi Kingdom that includes yeasts, moulds and mushrooms. Their characterization is essential  since all species have key ecological and economic roles; they nevertheless come up against methodologial obstacles. Metabarcoding is a method widely used today by biologists to easily identify microbes in an environment at low cost. The priniciple is the sequencing of a sufficiently discriminant genomic region that allows the identification of the different organisms in an environment.

However, the metabarcoding method up to now has not allowed to correctly use ITS (internal transcribed spacers) markers that are usually used to identify fungus species. The main difficulty with the ITS comes from the polymorphism of their size. The ITS amplicaons are DNA sequences obtained by PCR amplification. The problem is that the sequences thus amplified vary considerably in length. The metabarcoding method has until now been based on short reading sequences (two reads with 150 to 300 nucleotides for the same DNA fragment). Most tools treat only reads that overlap, only one of two reads or a combination of both. In all cases, the information from the sequencing is never entirely treated and a fraction of fungal species in the environment is never completely identified.

FROGS is an analysis tool using the metabarcoding technique that since 2015 has allowed the characterization of microbiota thanks to the treatment of marker amplicons, notably bacteria. It provides an innovative solution for the treatment of non-overlapping reads, for example the ITS, without the inconvenience of loss of information mentionned previously. FROGS is a fun tool, equipped with a graphic interface with the choice of using command lines or a web interface (Galaxy); the user can therefore rapidly and easily obtain a precise image of fungal communities present in the environments studied. FROGS produces very few falso positives or false negatives in comparison with the most popular tools that do not have this innovating read capacity (DADA2, QIIME2, USEARCH); it has an excellent sensitivity and high precision. 

This new version of FROGS is the result of a partnership between INRAE's GABI, MaIAGE (Jouy-en-Josas) and GenPHySE (Toulouse) laboratories. It is described in the scientific article published in Briefings in Bioinformatics and is available on the http://bioinfo.genotoul.fr/ and https://migale.inrae.fr/ platforms. FROGS can be used for many types of overlapping i.e. 16S, 18S, coi, rpob… or non-overlapping i.e. ITS1, ITS2, D1-D2, rpb2…amplicons.
FROGS codes are accessible and available on https://github.com/geraldinepascal/FROGS and via conda : https://anaconda.org/bioconda/frogs

For more information, visit the FROGS web site: http://frogs.toulouse.inrae.fr
Contact : frogs-support@inrae.fr