Depth of sequencing for host-associated mycobiome characterization

15 March 2019


The most popular NGS barcode currently used for fungal community identification is the internal transcribed spacer (ITS) region of the eukaryotic ribosomal cluster, which allows for species-level resolution in a large number of fungi.

However, the method suffers some inefficiencies, especially when investigating human-, animal- and plant-associated mycobiomes. The major problem encountered is that >99% of produced ITS sequences can originate from the host’s genome.

Even though molecular taxonomists are in continuous search to optimize primer pairs used for amplification (you can find some examples here, here and here) no significant progress has been made. What clearly emerges from those numerous attempts is that there is no universal solution, each study has its peculiarities and thus the optimal primer set performing slightly better.

Another inconveniency is that unlike the bacterial 16S sequencing, ITS library preparation protocol does not include reagents such as PNA PCR clamps, which block the amplification of contaminating host plastid sequences, and consequently increase yield.

It seems that at the moment not much can be done. The only solution is to increase sequencing depth in order to reach the minimum number of fungal reads/fragments that allows to capture heterogeneity of the community under investigation.

We’ve already discussed on the impact of sequencing coverage in metabarcoding experiments. Talking in numbers, we would suggest producing at least 1M of reads (instead of standard 50-100K) when analyzing eukaryotic host-associated fungal composition. The recommended depth agrees with sequencing efforts applied in a recent study on citrus rhizosphere microbiome.


Hope you've found some useful considerations, stay tuned for updates on metagenomics and other topics.

See you soon