Pinus Chloroplast Genomes HybSeq
Pinus Plastome Phylogenomics, HybSeq/Illumina sequencing
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 or 80 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.
Through next-generation sequencing, the amount of sequence data potentially available for phylogenetic analyses has increased exponentially in recent years. Simultaneously, the risk of incorporating ‘noisy’ data with misleading phylogenetic signal has also increased, and may disproportionately influence the topology of weakly supported nodes and lineages with rapid radiations and/or elevated rates of evolution. We investigated the influence of phylogenetic noise in large data sets by applying two fundamental strategies, variable site removal and long-branch exclusion, to the phylogenetic analysis of a full plastome alignment of 107 species of Pinus and six Pinaceae outgroups. While high overall phylogenetic resolution resulted from inclusion of all data, three historically recalcitrant nodes remained conflicted. Close investigation of these nodes revealed dramatically different responses to data removal. Whereas topological resolution and bootstrap support for two clades peaked with removal of highly variable sites, the third clade resolved most strongly when all sites were included. Similar trends were observed using long-branch exclusion, but patterns were neither as strong nor as clear. When compared to previous phylogenetic analyses of nuclear loci and morphological data, the most highly supported topologies seen in Pinus plastome analysis are consistent for the two clades gaining support from noise removal and long-branch exclusion, but inconsistent for the clade with highest support from the full data set. These results suggest that removal of noise in phylogenomic datasets can result not only in increased resolution for poorly supported nodes, but serve as a tool for identifying erroneous yet highly supported topologies. For Pinus chloroplast genomes, removal of variable sites appears to be more effective than long-branch exclusion for reducing the impact of noise.