title: Pinus Plastome Phylogenomics, HybSeq/Illumina sequencing
design: For plastome accessions novel to this study, total genomic DNA was extracted from frozen leaf or mega-gametophyte tissues using the FastDNA extraction protocol (MP Biomedicals, Ohio, USA). In several cases (Pinus chiapensis, P. cembroides, Pinus dabeshanensis, P. discolor, P. douglasiana, P. edulis, P. hwangshanensis, P. massoniana, P. pumila, and P. sabiniana), genomic DNA yield was insufficient for sequence preparation, so extracts were amplified by whole genome amplification with random hexamer priming (Pan et al. 2008). Genomic libraries were prepared following the Illumina protocol (Illumina 2007), with fragmentation performed using a BioRuptor Sonicator (Diagenode, Inc., Denville, NJ, USA) (setting ‘high’ for 5-30 one minute cycles). Adapters ligated to genomic fragments carried unique 4 bp ‘barcodes’ at their 3´ ends for multiplex sequencing as described in Cronn et al. (2008). Agarose gel size-selected (300-700 bp), adapter-ligated libraries were enriched through 12-18 cycles of PCR using Phusion DNA polymerase and HF Buffer (New England Biolabs, Ipswich, MA, USA) and standard Illumina paired-end primers, and quantified using a Nanodrop 1000 (ThermoFisher Scientific, Wilmington, DE, USA). Solution-based enrichment of the chloroplast portion of genomic libraries followed the general methods of Gnirke et al. (2009).     Libraries were submitted for sequencing singly or in barcode-specified multiplex pools ranging in size from four to 16 accessions. Most individual samples or multiplex pools were submitted to the Oregon State University Center for Gene Research and Biocomputing (OSU CGRB) (http://www.cgrb.oregonstate.edu/) for sequencing on the Illumina GAII sequencer, although several individual samples were submitted to the FAS Center for Systems Biology at Harvard University (http://sysbio.harvard.edu/csb/) for sequencing, also on an Illumina GAIIx sequencer. Libraries were loaded at a concentration of 5-7 ?M and sequenced in 60, 80 or 84 bp single-end sequencing reactions. Cluster formation, primer hybridization and sequencing reactions followed Illumina protocols (Illumina 2007). Image analysis, base-calling and error estimation were performed using the Illumina GA Pipeline version 1.5.