epiGBS of Sporobolus alterniflorus: SFB3R
We extracted genomic DNA (gDNA) from the 300 samples using the Qiagen DNeasy plant mini kit according to the manufacturers protocol with some modifications (sensu Foust et al., 2016). We prepared epiGBS libraries for 288 samples using three sets of 96 barcodes to be sequenced on three lanes of the Illumina Hi-Seq platform following methods outlined in (van Gurp et al., 2016) and modified in Boquete et al., (Boquete et al., 2020). We digested 400 ng of genomic DNA from each sample with the enzymes NsiI and AseI. We ligated methylated, nonphosphorylated, barcoded adapters with variable barcodes of lengths from 4 to 6 base pairs to either end of the resulting fragments. The barcoded adaptors were designed so that we could identify forward (Watson) and reverse (Crick) strands for each fragment within each individual. Having the strand information allows for differentiating between C/T polymorphisms and methylation polymorphisms because we can recreate when unmethylated cytosines were present in either strand before bisulfite treatment (for details see van Gurp et al., 2016). For each group of 96 samples, we allocated the samples across 8 pools and cleaned and size-selected gDNA fragments greater than 200 bp using 0.8 SPRI beads (Agencourt AMPure XP; Beckman Coulter). We repaired nicks between the 3 fragment overhangs and the 5 non-phosphorylated adaptor nucleotides using a mix of dNTPs and DNA polymerase I. We used the Zymo EZ Lightning methylation kit to bisulfite-treat the DNA. We amplified libraries with the KAPA Uracil Hotstart Ready Mix with the following PCR conditions: an initial denaturation step at 95C for 3 min followed by 18 cycles of 98C for 10s, 65C for 15s, and 72C for 15s, with a final extension of 72C for 5 min. The University of Florida Interdisciplinary Center for Biotechnology Research (UF ICBR) sequenced pairedend reads of the libraries on three lanes of the Illumina Platform HiSeq3000 (2  150 bp) in March 2017. Due to poor sequencing quality and depth for many of the samples, we repeated library preparations with slight modifications to improve library quality for the 288 samples and an additional 5 samples for a final total of 293 samples. We were unable to obtain additional high quality DNA for the remaining seven samples and they were not included in the final design. We meta-pooled samples following ligation to reduce heterogeneity during the remaining library preparation process. The samples were sequenced at the Novogene facility in Hong Kong on four lanes of the Illumina Platform HiSeq X Ten System (2  150 bp) in January 2019. We found that there were significantly fewer average reads per lane in the first set of libraries than the second set (P < 10-16), but we could not detect significant differences in the overall read count between populations, habitats or their interaction with the two library preparations (Figure S.1). We combined the sequencing from all seven lanes of data for analysis.