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.