Computational protocol: Rapid and accurate pyrosequencing of angiosperm plastid genomes

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Protocol publication

[…] Fresh leaf material of Nandina and Platanus was collected on the campus of the University of Florida for chloroplast isolation. Voucher specimens (Nandina, M. J. Moore 310; Platanus, M. J. Moore 309) have been deposited in the herbarium of the Florida Museum of Natural History (FLAS). Purified chloroplasts were isolated from approximately 8.2 g of Nandina leaf material and 30.8 g of Platanus leaf material, following the sucrose gradient protocol of Jansen et al. []. Two 25-mL sucrose step gradients were used for each species. The purified chloroplasts were lysed in a solution containing 1.0 μL chloroplasts, 4.0 μL 1× PBS, and 1.5 μL activated solution A []. The lysis reactions were incubated on ice for 10 min, and then were stopped using 3.5 μL of solution B []. The released chloroplast DNA (cpDNA) was amplified via rolling circle amplification (RCA) [] using the Repli-G kit (Qiagen, Inc., Valencia, CA, USA), following the manufacturer's instructions. To assess the relative percentage of cpDNA vs. nuclear DNA, RCA products were digested with EcoRI and visualized on agarose gels following the protocol in Jansen et al. [].GS 20 library construction and sequencing were performed as described in the supplementary material and methods of Margulies et al. [] with slight modifications as specified by 454 Life Sciences. Briefly, high molecular weight DNA from the RCA reactions was sheared by nebulization to a size range of 300–800 bp. DNA fragment ends were repaired and phosphorylated using T4 DNA polymerase and T4 polynucleotide kinase. Adaptor oligonucleotides "A" and "B" supplied with the 454 Life Sciences sequencing reagent kit were ligated to the DNA fragments using T4 DNA ligase. Purified DNA fragments were hybridized to DNA capture beads and clonally amplified by emulsion PCR (emPCR). DNA capture beads containing amplified DNA were deposited onto a 40 × 75 mm PicoTiterPlate equipped with an eight-lane gasket. This gasket divides the plate into eight identical regions (lanes) in which the pyrosequencing reactions occur during GS 20 sequencing runs. Each species was initially assigned four lanes on a single plate for a titration sequencing run, which is a standard preliminary sequencing run in which the relative quality of GS 20 libraries is assessed. Preliminary analyses of these data allowed for the estimation of the number of additional GS 20 sequencing lanes (three for Nandina, five for Platanus) on a second plate that were necessary to obtain approximately 20× coverage for each genome. This second sequencing run will be referred to as the supplementary run.DNA sequence data from the titration and supplementary runs were combined in a single assembly for each species using version 1.0.52.06 of the GS 20 Newbler sequence assembly software. These data are referred to as the combined run data. The combined data contigs were then imported into DOGMA [] to determine their approximate positions within the plastid genome. Based on this information, putatively adjacent contigs were examined in Sequencher 4.2 (GeneCodes Corp., Ann Arbor, MI, USA) in order to unite any contigs where short sequence overlap at the ends went undetected in the initial assembly. Gaps between contigs were bridged by designing custom primers near the ends of the GS 20 contigs for PCR and conventional capillary-based sequencing.To estimate the accuracy of the GS 20 sequence, custom primers were designed to check all possible frame shift errors encountered in the preliminary DOGMA annotation of the GS 20 sequence of both genomes using PCR and conventional sequencing. In addition, the four junctions between the inverted repeat (IR) and single-copy (SC) regions of both genomes were sequenced conventionally, as was the entire IR region for both genomes. The IR regions were amplified using the recently described ASAP method [], which utilizes a set of 27 overlapping primer pairs that are designed to obtain extensive coverage of the IR across the phylogenetic diversity of angiosperms. RCA product derived from the same chloroplast isolations used in GS 20 sequencing was utilized for all amplifications involving the single-copy regions of Nandina and for all regions of Platanus. The IR region of Nandina was amplified from a separate total DNA isolation from a different individual collected at Kanapaha Botanical Gardens in Gainesville, Florida (A. Dhingra s.n.).The completed genome sequences were annotated using DOGMA and are available in GenBank. […]

Pipeline specifications

Software tools Newbler, DOGMA, Sequencher
Applications Genome annotation, Phylogenetics
Organisms Nandina domestica, Platanus occidentalis
Chemicals Nucleotides