 Solution

Pooling can erase between-replicate variation. One unusually well-covered or highly modified sample can dominate the pooled estimate, hiding an inconsistent biological response. If individual reads are then treated as independent biological replicates, uncertainty can be understated through pseudoreplication.

A better analysis retains replicate identity and models sample-to-sample variation, while allowing information to be shared about suitable common component parameters. Pooled performance on a larger dataset can be useful, but should not substitute for evidence that the result reproduces across independent biological samples.