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.