Laboratory work
First, we tested in singleplex reactions for the locus-specificity of
selected primers and their universal applicability to catarrhine species
in a panel representing all major lineages of catarrhines (Table 1).
High-quality DNA from a male of each of the ten species was obtained
from the Gene Bank of Primates at the German Primate Center. PCRs were
performed in total volumes of 25 µl containing 1x QIAGEN Multiplex PCR
Master Mix (QIAGEN), 0.4 µM of each primer and 50 ng genomic DNA.
Cycling conditions comprised of 15 min at 95°C, 30 cycles each with
denaturation at 94°C for 30 sec, annealing at 57°C for 90 sec and
extension at 72°C for 90 sec, and a final extension step of 10 min at
72°C. All reactions were run together with no-template controls (NTCs)
to check for contamination. PCR performance was checked on 2% agarose
gels stained with ethidium bromide (Carl Roth GmbH). Sequencing of
singleplex PCR reactions was omitted.
Next, we tested for the possibility of running multiplex PCR reactions
to reduce overall laboratory work and costs. Therefore, we pooled either
all 45 primer pairs in a single PCR reaction (1-pool approach) or
divided them into five PCR reactions each containing nine primer pairs
(5-pool approach) or three PCR reactions containing 18 and 2 x 12 primer
pairs (3-pool approach; for rationale of pooling and locus exclusion see
Results). Amplifications were conducted as described for the singleplex
PCRs (same PCR set-up, DNA samples, cycling conditions, NTCs), but with
different primer concentrations (see Tables S3-S5 for pooling schemes
and concentrations of single primers within pools). To minimize PCR
errors, we ran PCR reactions in two independent replicates. PCR
performance was again checked on 2% agarose gels. Replicate PCR
products (including the NTCs) were pooled and then cleaned with the
MinElute PCR Purification Kit (QIAGEN). DNA concentrations were measured
with a Qubit 3.0 (ThermoFisher Scientific) and 100 ng were subjected to
indexing PCR. Indexing PCR was performed in total volumes of 25 µl
containing 1x KAPA HiFi HotStart ReadyMix (Roche), 0.4 µM of each
indexing primer and 100 ng purified PCR product. Cycling conditions
comprised of 45 sec at 98°C, 4 cycles each with denaturation at 98°C for
15 sec, annealing at 62°C for 30 sec and extension at 72°C for 30 sec,
and a final extension step of 1 min at 72°C. Subsequently, indexed PCR
products were purified with the MinElute PCR Purification Kit (QIAGEN)
and ran on a Bioanalyzer 2100 (AGILENT) to check for PCR performance and
molarity. Libraries were diluted to a final concentration of 10 nM and
then pooled and sequenced with 51 cycles forward and 251 cycles reverse
on Illumina’s MiSeq desktop sequencer.
To check for Mendelian inheritance and whether our new microsatellite
panel is also applicable to low-quality and -quantity DNA as typically
extracted from fecal samples (Monteiro et al., 1997; Perry, Marioni,
Melsted, & Gilad, 2010), we tested our panel in 12 fecal samples of
wild Guinea baboons. The samples comprised of six males and two
‘families’ each composed of a male, a female and their known offspring.
DNA from these 12 specimens was previously genotyped via FLA at 24
microsatellite loci (Dal Pesco, 2019). The amplification procedure and
follow-up steps for the applied 3-pool approach were the same as
described above, but the number of cycles in the initial amplification
was increased to 40, the total DNA amount was increased to 200 ng, and
each PCR was performed in triplicates.