Nebnext library prep kit

We used 500 ng of total DNA per sample to prepare the genomic libraries. DNA fragments of ca. 250 bp were size‐selected with a bioruptor sonicator and the length distribution of fragments was evaluated by automated electrophoresis using a 2100 Bioanalyzer System (Agilent, Santa Clara, California, USA). Barcode adapters were ligated for Illumina sequencing using a NEBNext library prep kit for three samples (P. lagunae BS3‐BS5) and a TruSeq library prep kit for all other samples (Illumina, San Diego, California, USA).
Pools of 24 samples were enriched for nuclear targets with MYbaits version 2.3.1 biotinylated RNA baits (Arbor Biosciences, Ann Arbor, Michigan, USA) following the manufacturer's protocol. Probes were designed for 1045 putative low‐copy nuclear genes from Pinus taeda L. (Willyard et al., 2007 (link); Neves et al., 2013 ; Gernandt et al., 2018 (link); see Appendix S2 for more details). The samples were spread across eight sequencing runs (Appendix S3) that also included Pinaceae samples for other studies. Different mixtures of enriched and unenriched libraries were used for successive runs, according to recovery of plastid sequences in prior runs. We combined samples into three different multiplex sets (Appendix S3) to sequence on a single lane each of an Illumina Hi‐Seq. 2500 or Hi‐Seq. 4000 using the 100, 125, or 150 bp modules with paired reads (Appendix S3).