The most convenient way to barcode a sample is the use of barcoded adapters, as depicted in the barcode design overview. One minor disadvantage is that the ligation might not be as efficient as with standard SMRTbell adapters, leaving some molecules only with one adapter. As barcoded adapter designs are inherently symmetric, we implemented ways to recover the demultiplexed yield from one-sided barcoded molecules with ease, using
As the first step, generate HiFi data with ccs v6.3.0 or later. This version will store additional tags per records, indicating if the molecule has missing adapters on either side. The second step is to use the new
--hifi-preset SYMMETRIC-ADAPTERS introduced with lima v2.5.0, described here. That’s it. All of this done automatically starting with Revio.
--single-end was introduced with Revio in SMRT Link v12.0 and lima v2.7.1. It allows to only score one side of a HiFi read, if the other flank is molecularly missing a barcoded adapter. It is controlled by
--min-end-score and is set by default for both SYMMETRIC HiFi presets. If you see a barcode not matching one side of the HiFi read at all, check the
ac tags to see if the read is missing the read is missing the barcoded adapter on one side and the
--single-end heuristic got activated. In lima v2.9.0 we improved this option to further reduce barcode miscalls.