Demultiplexing Twist 96-Plex libraries differs from the standard process. The individual sample barcodes are in line as a part of R1, instead of in the standard i5 location. Because of this, Illumina’s bcl2fastq2 and BCLconvert tools do not recognize the individual sample barcode and instead combine all wells together into a single plate-level FASTQ file during demultiplexing, based on the single i7 index. 

To address this, 96-Plex users must follow a two step demux process. First, the per-cycle BCL files must be converted into a plate-level FASTQ, and then followed by a second conversion of the plate-level FASTQ files into sample-specific FASTQ files. We recommend using the fgbio DemuxFastqs open source software to produce per-sample FASTQs. See the demultiplexing guide on the Twist website for more information.