Library Preparation
In order to evaluate the variation of resulting genotypes, individual PCR replicates required library preparation for Illumina sequencing. Individual dual iTru indices (Glenn et al., 2019) were ligated to each PCR replicate using KAPA Illumina Library Preparation Kits (Roche, # KK8232) following the reduced reactions previously published (Hawkins, Leonard, et al., 2016). Additionally, all PCR replicates across all microsatellites were pooled together for another separate library preparation in order to determine if pooling across replicates influenced resulting genotypes (hereafter referred to as the pooled dataset). Two µl of each PCR replicate (mitochondrial DNA and microsatellites) was pooled from museum specimens prior to library preparation, and 2 or 3 µl from tissue replicates of mitochondrial DNA and microsatellites respectively. A library preparation control (consisting of ddH20) was included in all steps. Libraries, as well as a negative control, were amplified in 25 µl reactions consisting of 1.25 µl of each iTru adapter, 2.5 µl ddH2O, 7.5 µl of adapter-ligated DNA, and 12.5 µl of KAPA HiFi HotStart ReadyMix. The thermocycler conditions for library amplification were: 98°C for 45 sec; 10 cycles (tissue sample) or 14 cycles (museum samples) of 98°C for 15 sec, 60°C for 30 sec, 72°C for 1 min; 72°C for 5 mins. After completing library prep, an agarose gel was run on all replicates to ensure successful ligation of Illumina and iTru adapters. Products were SPRI cleaned as detailed above. Each individually prepared replicate was then pooled together by placing 2 ul of cleaned product into a 0.5 mL tube, which was quantified for Illumina sequencing on an ABI QuantStudio 3 using the KAPA Biosystems Library Quantification Kit (Roche, # KK4824), standard Illumina Primers and following the protocol therein.