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How to barcode your sample

Lima can demultiplex samples that have an unique per-sample barcode pair and have been pooled and sequenced on the same SMRT cell. There are four different methods to associate barcodes with a sample, by PCR or ligation:

  1. Sequence-specific primers
  2. Barcoded universal primers
  3. Barcoded adapters
  4. Probe-based linear barcoded adapters

How to combine barcode sequences

In addition, there are three different barcode library designs. In order to describe a barcode library design, one can view it from a SMRTbell or read perspective. As lima supports CLR subread and CCS read demultiplexing, the following terminology is based on the per (sub-)read view.

In the overview above, the input sequence is flanked by adapters on both sides. The bases adjacent to an adapter are referred to as barcode regions. A read can have up to two barcode regions, leading and trailing. Either or both adapters can be missing and consequently the leading and/or trailing region is not being called.

For the symmetric and tailed library design, the same barcode is attached to both sides of the insert sequence of interest; the only difference is the orientation of the trailing barcode. For identification, one read with a single barcode region is sufficient.

For the asymmetric design, a different barcode pair is attached to the sides of the insert sequence of interest. In order to be able to identify a different barcode pair, a read with leading and trailing barcode regions is required.

Output barcode pairs are generated from the identified barcodes. The barcode names are combined using the -- infix, for example bc1002--bc1054. The sort order is defined by the barcode indices, lowest first.


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