Hi @chuang!
We usually see this error when there was an overlap-related issue when joining your paired-end reads. The ability for reads to be joined successfully is dependent on a handful of factors, including read quality, truncation settings, and adapter removal, to name a few. Have you made sure your sequencing adapters are removed? If so, what does your demux summarize quality plot look like? Please attach here (or a screenshot), this will help us understand why you picked the truncation settings that you provided above. Thanks! 