DNA extracts from blood and ear tissue samples from Wadi Sareen were assessed for DNA quality via agarose gel electrophoresis on a 2% gel, and 21 samples (TAH044-55, TAH057-59 and TAH060-64) with suitable non-degraded DNA selected for the library preparation stage. DNA was quantified using a Qubit Assay (Thermofisher Scientific) and normalised to 7 ng–1 µl.
A double digest RAD (ddRAD) library was produced according to a modified protocol of Peterson et al. (2012 (link)) (see also Bourgeois et al. (2018 (link)) for the modified protocol) using the restriction enzymes SphI and SbfI and a gel excision section of 320–590 bp following restriction of the DNA with the two enzymes. Individuals in the library were barcoded using a barcode on each end of the fragment (double barcode combination) and each individual was repeated twice with a different barcode across the library to improve evenness of coverage. The barcoding system allows DNA from multiple individuals to be “tagged” and pooled into a single lane of sequencing. An additional positive control was included to allow for quality control of the experimental process and for assessment of genotyping error-by-read depth. The resulting library was quantified using a Qubit Assay (Thermofisher Scientific) and then sequenced in both directions (Read 1 and Read 2) using a single lane of MiSeq Sequencing Technology (Illumina).
A double digest RAD (ddRAD) library was produced according to a modified protocol of Peterson et al. (2012 (link)) (see also Bourgeois et al. (2018 (link)) for the modified protocol) using the restriction enzymes SphI and SbfI and a gel excision section of 320–590 bp following restriction of the DNA with the two enzymes. Individuals in the library were barcoded using a barcode on each end of the fragment (double barcode combination) and each individual was repeated twice with a different barcode across the library to improve evenness of coverage. The barcoding system allows DNA from multiple individuals to be “tagged” and pooled into a single lane of sequencing. An additional positive control was included to allow for quality control of the experimental process and for assessment of genotyping error-by-read depth. The resulting library was quantified using a Qubit Assay (Thermofisher Scientific) and then sequenced in both directions (Read 1 and Read 2) using a single lane of MiSeq Sequencing Technology (Illumina).
Free full text: Click here
