Fig. 1

Mechanism for index hopping and its effects on sequencing library demultiplexing. a-e Illustration of index hopping due to (a) free adapter molecules remaining after purification post-PCR, resulting in (b) mis-priming of a single stranded library molecule. c The mis-primed library molecule is extended via ExAmp polymerase to generate (d) a fully complete library molecule with an incorrect sample index assigned. e Both correct and index-hopped molecule can form clusters on the flow cell. f-i Demultiplexing runs with single- or dual-indexed libraries with index hopping. f The case with a single index and no index hopping where the read(s) for a cluster are associated with a specific sample index (green with green and blue with blue) added to each molecule during library preparation, allowing reads to be assigned to its correct library of origin. g The case as above but with index hopping (a blue index now marks a green cluster), where that read will be incorrectly assigned to the wrong library. h A unique dual-indexed strategy allows for a single sample to have 2 indexes to be associated with a single library molecule. Here, library 1 = yellow + green, library 2 = purple and blue. i The case as above but with index hopping will result in reads displaying unanticipated combination of indexes (e.g., purple + green). The reads associated with unanticipated indexes can then be filtered out