Nanopore sequencing of quantitative PCR assays reveals off target Aspergillus species commonly found in Cannabis flowers

Illumina Sequecing of a qPCR assay was performed. Paired Reads were merged with FLASH, trimmed and q30 quality filtered and BLAST'd against NCBI NR. Krona plots were generated with the top 1000 reads. 

For ONT analysis, 2 different ITS amplicons (450bp and 750bp) were amplified from the Pre-PCR DNA to assess the microbiome diversity the qPCR assay was targeting. 

The 450bp ITS assay is fungal specific. 

The 750bp ITS assay include plants and fungi (Cheng et al). 

Amplicons were converted to Barcode ready sequencing libraries and sequenced on a Flongle with SUP basecalling mode. Reads >Q14 and 200-800bp in length were BLAST'd and fed into Krona plots.