Could you send me a link to your input file? All the testing for ITSxpress was done with data imported in PairedEndFastqManifestPhred33 format not CasavaOneEightSingleLanePerSampleDirFmt. I think ITSxpress may be making some incorrect assumptions about the structure of the input data. This issue came up a second time here Trimming fungal demultiplexed paired-end sequences before dada2.