Bioinformatic analysis
The data analysis was performed using the software package CHIIMP
v.3.0.0 (Barbian et al. 2018). The raw data (FASTQ files) as well as all
input files (config-file, sample-file, locus-attributes-file) are
available in the online supplement resources. As our microsatellite
panel included several di-repeat loci, which stutter more frequently
than tetra-repeats, we increased the stutter count ratio to 0.70
(stutter.count.ratio_max: 0.7). We further implemented a broad range of
possible allele lengths in the locus attributes by setting the length
buffer to 100 bp. This ensured the inclusion of all tested species even
if the allele sizes at a given locus varied between species according to
the available reference genomes. The minimum number of reads per locus
was set to 100 (counts.min: 100). All other parameters were set to
default.
With the current version of CHIIMP, wobble positions in primer sequences
cannot be accounted for. Hence, for loci with a wobble position in a
primer sequence, alternative nucleotides of the wobble are erroneously
recognized as different alleles. Moreover, the repeat motif needs to be
specified in CHIIMP, but as repeat motifs can vary in the investigated
species, correct (orthologous) reads remain unrecognized for some
species if CHIIMP is fed with a wrong repeat motif. Due to these
reasons, the output for all loci was checked manually and corrected if
needed. Additionally, we screened the processed reads for the general
level of amplification per locus and the occurrence of PCR artifacts
(off target amplification, primer dimer, false primer pairings, etc.).