Fungal ITS Mock Community Spike-in Control

We constructed a spike-in mock community control for use in next-generation sequencing studies of the fungal internal transcribed spacer (ITS) region which consists of cloned ITS sequences that vary in length, GC content, number of homopolymer stretches, as well as taxonomic classification. Using this positive control, we show that the primary DNA sequence and the initial PCR reaction strongly bias results, indicating that abundance metrics are not representative of the fungal community even in an extreme example of equally mixed plasmids. Moreover, we demonstrate the utility of using the spike-in mock community for fine-tuning downstream software parameters and validating datasets on both the Ion Torrent Personal Genome Machine (PGM) and the Illumina MiSeq platforms.