Hi @LuSanto - you are now describing a separate, and what I believe to be, unrelated, issue. The primary issue in this post is that your forward barcodes are not identifiable in your reads. The secondary issue is the one you just posted, about only the first sample in a group of matching forward barcodes being demultiplexed. I will take a closer look at the secondary issue, but the primary issue can only be resolved by you and/or your sequencing center --- you need to make sure that the orientation of reads match that of the barcodes. The fact that you are getting such low recovery is a smoking-gun that there is an issue here.