Mycobiome Metagenomics with Element LoopSeq™

Single molecule sequencing of the complete ~2.5 kb 18S-ITS1-5.8S-ITS2 region

Uncover the complexity of fungal communities with full-length sequencing the complete 18S-ITS1-ITS2 region

Occupying an incredible diversity of ecological niches, fungi play many important roles including as decomposers, nutrient transporters and recyclers, pathogens, symbionts, and commensals. Yet only a small fraction of fungal species―estimated to total anywhere from 1.5 million to 12 million―have been formally characterized. Even potentially medically relevant species living in fungal communities within the human body are not well-identified.

One of the big problems has been fungal culture, as many species do not thrive under common culture conditions. Another issue is that culture-independent identification methods have not been of sufficiently high resolution to provide species-level taxonomic assignments.

While high-throughput sequencing methods are revolutionizing biology, studies of fungal communities have not kept up. Finding genomic regions that are short enough for standard NGS approaches but variable enough to distinguish one species from another has been challenging. Researchers often have to choose between sequencing portions of the 18S ribosomal RNA gene, the internal transcribed spacer 1 (ITS1), or the internal transcribed spacer 2 (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.

But what if you could sequence the entire 18S-ITS1-ITS2 region?

It's time to find out.

Maximize classification power by obtaining full-length sequence of individual amplicons.

Obtain greater insight into the composition of fungal communities with improved accuracy of abundance estimates and taxonomic determination

The challenge with conventional next generation sequencing (NGS) approaches to fungal metagenomics studies has been finding genomic regions that are short enough for NGS but variable enough to distinguish one species from another. Researchers often have to choose between sequencing portions of the 18S ribosomal RNA gene, the internal transcribed spacer 1 (ITS1), or the internal transcribed spacer 2 (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 Mycobiome Kit, the vast majority of reads are assembled into the full-length 18S-ITS1-ITS2 sequence (Figure 2). This can be easily seen in Figure 2A, where reads from a synthetic community composed of two species assemble into two main peaks―one peak for the full-length 18S-ITS1-ITS2 region from Saccharomyces cerevisiae and the other for the full-length 18S-ITS1-ITS2 region from Cryptococcus neoformans. When applied to a soil sample containing a complex and unknown number of diverse fungal species (Figure 2B), the distribution of read lengths reflects the complexity of the sample with a main peak around 2,400 bp, a minor peak around 2,800 bp, and a number of reads of different lengths suggesting sequence from a range of organisms.

LoopSeq’s longer read-length directly translates into more accurate taxonomic assignment (Figure 3). Because LoopSeq synthetic long-read sequencing involves generating short reads which are then reassembled in silico into the parent molecule (turn the page to learn more about the workflow), we can easily assess the accuracy of LoopSeq’s taxonomic assignments by comparing unassembled short-reads with the synthetic long-reads.

Using a defined mock community consisting of an equal number of four fungal species, only 48% of the unassembled short reads could correctly identify organisms at the genus level and 26% at the species level (Figure 3). In contrast, when these same short-reads were assembled into the full-length synthetic long-reads, more organisms were correctly identified at a higher taxonomic resolution, with 93% of contigs larger than 1 kb able to provide correct identification at the genus level and 66% at the species level.

With longer stretches of highly accurate rDNA sequence, the LoopSeq 18S-ITS1-ITS2 Mycobiome Kit is ready to help researchers unravel the complexities of the mycobiome and better understand how this large and incredibly diverse group of organisms contributes to ecosystem functioning, whether that’s within a specific tissue or across entire biomes.

Eliminate PCR bias and improve sequence accuracy with single molecule counting and consensus error correction

Supercharge discovery with contigs queried against three major databases.

• EnsemblFungi
• Unite Community
• Silva High-Quality Ribosomal RNA Databases

Discover with confidence using validated kits and services

As with all of our kits, the 18S-ITS1-ITS2 LoopSeq Mycobiome Kit has been validated using several mock communities. We recovered operational taxonomic units (OTUs) belonging to all members in the different mock communities that we tested, despite DNA abundances spanning 3 orders of magnitude in some of the communities, and taxa were consistently on target.