DNA-based identification of AM fungi from environmental samples
General Philosophy - Research in our lab is interested in all fungi, but particularly focused on AM fungi. This results in methodological challenges in the DNA-based identification of fungi in environmental samples. Although the use of the internal transcribed spacer (ITS) regions ITS 1 and/or ITS2 are generally considered appropriate for species-level differentiation among most fungal taxa, there is no consensus regarding the best amplicon rRNA gene region for the species-level identification of AM fungi. Different research groups working on AM fungi have different preferred regions among the ITS, the small subunit (SSU), and the large subunit (LSU) regions. There are many pros and cons for using these different regions such as efficacy and reliability of primers, the quality of available databases for taxon assignment, but most importantly, differing rates of AM fungal evolution among regions which results in variable ability to resolve sequences at the species level (Egan et al. 2018, Lekberg et al. 2018). The Chaudhary Lab has settled on the use of SSU as our preferred rRNA gene region for the identification of AM fungi because it provides significantly better species-level resolution than ITS and has a curated, more accessible public database (i.e. MaarjAM) for taxon assignment than LSU. We made significant investments in this approach prior to the publication of the Deleveaux et al. 2020 LSU region database, which looks promising.
These are difficult methodological decisions to make and we expect changes to this approach in the future. For example, long-read sequencing technologies like Oxford nanopore or Pacbio have the potential to solve the problem of which gene region to choose because they are able to sequence the entire rRNA region in one step. These methods are still in the developmental phase for mixed environmental samples, but the current status is that long-read technologies don’t improve species resolution, have a higher error rate, don’t yet have solid bioinformatics pipelines, and haven’t yet been confirmed using mock community studies. Still, we are optimistically awaiting a time when disagreement over the best amplicon region for AM fungi ends and are staying apprised of new methods to employ them as they become more reliable.
These are difficult methodological decisions to make and we expect changes to this approach in the future. For example, long-read sequencing technologies like Oxford nanopore or Pacbio have the potential to solve the problem of which gene region to choose because they are able to sequence the entire rRNA region in one step. These methods are still in the developmental phase for mixed environmental samples, but the current status is that long-read technologies don’t improve species resolution, have a higher error rate, don’t yet have solid bioinformatics pipelines, and haven’t yet been confirmed using mock community studies. Still, we are optimistically awaiting a time when disagreement over the best amplicon region for AM fungi ends and are staying apprised of new methods to employ them as they become more reliable.
Environmental aerial fungal sampling
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Lab Standard Operating Procedures for MWAC (passive dust collector) Installation, Spring Reset, and Fall Sampling can be downloaded here:
https://github.com/thebalachaudhary/flyingfungi
https://github.com/thebalachaudhary/flyingfungi