How chimeras reads generated in PacBio?

Question

[Medhat]

As it is declared in my question, I would like to know if anyone have an idea how chimera formed in SMRT PacBio sequencing?

here Thomas Hackl try to explain it; but I would like to have more explanation. also what is siamaeras?

Answer 1

[rhall]

SMRT Bells are blunt end ligated to the DNA fragment being sequenced, even if they are in an excess there is a non-zero chance of a DNA fragment ligating to another DNA fragment before having a SMRT Bell adapter added. This would generate the classic chimera, a sequenced read being from two random parts of a genome. Note this is random and will not happen at exactly the same location more than once, so it is easily dealt with at the analysis stage. The "siamaeras" or missing adapter look like a sequence followed by the reverse complement of the sequence, and results from the SMRT Bell being missing / not-detected on one end of the insert i.e. you read a sequence forward then backwards without an adapter so the software does not know that the read needs to be split. This can happen due to sample prep - a long overhang forms a hairpin, mimicking a SMRT Bell, or less frequently due to a real SMRT bell being missed in software. See the template prep guide for more info