  Single-stranded libraries generated from ancient DNA extracts have specific
sequencing requirements. The short library molecules typically associated with
ancient DNA templates are expected to behave differently during the annealing
to the flow cell and subsequent cluster generation (bridge PCR), requiring
optimisation of loading amounts to obtain optimal and consistent cluster
densities. For the past 3 years, we have carried out sequencing of
single-stranded libraries on the Illumina NextSeq 500 sequencing platform at
the Institute for Biochemistry and Biology, University of Potsdam. We report
our optimisations here. This document may be useful for other researchers
wishing to sequence single-stranded libraries on the NextSeq 500 platform. It
does not replace the excellent documentation provided by Illumina (links
provided below), but rather serves as additional information specific to
single-stranded libraries. The original papers describing the library protocol
should also be studied in detail, and complement the information presented
here.
