We ran Lexogen’s QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina on RNA of 49 multiplexed human samples in 4 lanes, barcoded with i7 Lexogen’s indexes, in NextSeq550. During demultiplexing with bcl2fastq, as provided by Illumina on the NextSeq550 and with the information for the sample sheet extracted from Lexogen’s i7 indexes, many reads (almost ~49% of the reads, uniformly distributed in all 4 lanes) were returned as “undetermined”, appended in 4 different fastq files (one for each lane). Has anybody else run into such a situation before? If yes, any idea why this is happening and if it is possible to demultiplex those reads, when bcl2fastq cannot demultiplex them? I have not run any code for the bcl2fastq. The software was already installed in the Nextseq550 machine and the demultiplexing was applied by the machine.
Additional data information can be provided but I am not sure what else I should provide here without any hint.
Thank you very much,