While High-Throughput sequencing methods have been revolutionizing Biology, studies of fungal communities have not kept up with this pace.
The big challenge here is to find genomic regions that are short enough for standard NGS approaches, but variable enough to distinguish one species from another. Often, researchers do have to choose between sequencing portions of the 18S ribosomal RNA gene, the internal transcribed spacer 1 (ITS1) or ITS2. However, each of these regions possesses unique taxonomic biases, and none of them are sufficient, on their own, for accurate identification at the species level and, in some cases, at the genus level.
With the LoopSeq technology, it is possible to cover the entire 18S ribosomal RNA gene and both ITS1 and ITS2. This enables accurate classification and quantification, compared to the classical Illumina workflows.
The LoopSeq Mycobiome kit enables assembly of the vast majority of reads into the full length 18S-ITS1/2 sequence. The figure on the left shows the reads of a synthetic community, composed of 2 species, being assembled into 2 main peaks. One peak for the full length 18S-ITS1/2 region of Saccharomyces cerevisiae and the other for the full length 18S-ITS1/2 region of Cryptococcus neoformans.
When applied to a soil sample containing a complex and unknown number of diverse fungal species (figure on the right), the distribution of the read lengths reflects the complexity of the sample with a mean peak around 2,4Kb, a minor peak around 2,8Kb and a number of reads of different lengths suggesting sequence from a range of organisms.