P.teada BAC project
The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences
Whole genome shotgun sequencing was performed on diploid DNA from the same individual used to construct the BAC library, using the high-throughput Illumina Genome Analyzer II sequencing platform. Genomic DNA library construction was carried out using the Illumina genomic DNA sample preparation kit according to manufacturer’s instructions, except that paired end specific oligonucleotides were used instead of the single read oligonucleotides. Starting material was 80 ul of pine genomic DNA at a concentration of 62.5 ng/ul sonicated in a Diagenode Bioruptor for 15 cycles of 30” on maximum power then 30” rest.  Following paired end adapter ligation, fragments of approximate size 400-425 bp were gel purified and PCR amplified using the paired end Illumina library PCR primers (primers 1.0 and 2.0).  After AMPure purification (Beckman Coulter Genomics), the sample was applied to an Agilent Bioanalyzer for quantitation.  Based on the bioanalyzer-reported sample concentration, the library was applied to a flow cell at 5 pM using v1 cluster reagents.  Sequencing was performed on an Illumina Genome Analyzer II using version 2 sequencing reagents for 40, 42 and 60 cycles. Basecalling was carried out using the Illumina GA Pipeline v 1.3. The WGS sequencing was carried out at the University of California, Davis, Genome Center.
In today’s age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but we have little information on the nature and identity of repetitive units in gymnosperms. The pines (Pinus) have extensive genetic resources, with approximately 329,000 ESTs from eleven species and genetic maps in 8 species, including a dense map of the twelve linkage groups in Pinus taeda. We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (= 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. This study indicates that the majority of repeats in the P. taeda genome are ‘novel’ and will therefore require additional BAC or genomic sequencing for accurate characterization. This study provides the first evidence that sequencing a pine genome using a WGS approach is a feasible, and highly advantageous, goal.