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Why are different barcode pair hits reported in –same mode?

Lima tries all barcode combinations and --same only filters BAM output. Sequences flanked by different barcodes are still reported, but are not written to BAM. By not enforcing only same barcode pairs, lima gains higher PPV, as your sample might be contaminated and contains unwanted barcode pairs; instead of enforcing one same pair, lima rather filters such sequences. Every symmetric / tailed library contains few asymmetric templates. If many different templates are called, your library preparation might be bad.

Why are same barcode pair hits reported in the default different mode?

Even if your sample is labeled asymmetric, same hits are simply sequences flanked by the same barcode ID.

But my design does not include same barcode pairs! We are aware of this, but it happens that some ZMWs do not have sufficient signal to call a pair with different barcodes.

What are undesired hybrids?

When running with --peek-guess or similar manual option combination and different barcode pairs are found during peek, the full chip may contain low-abundant different barcode pairs that were identified during peek individually, but not as a pair. Those unwanted barcode pairs are called hybrids in lima.


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