454 sequencing of ginseng transcriptome
A cDNA library was constructed from isolated mRNAs using a ZAP-cDNA Synthesis Kit (Stratagene, Santa Clara, CA, USA) following the manufacturer's protocol and then cDNA was fragmented by nebulization for library construction. For 454 pyrosequencing, the GS FLX General Library Preparation Method Manual workflow (Roche Diagnostics) was followed. First-strand cDNA synthesis was performed with oligo(dT) primer as described in the provided protocol using 500 ng total RNA. Then 454 sequencing adapters A (5’-GCCTCCCTCGCGCCATCAG-3’) and B (5’-CTGAGCGGGCTGGCAAG-GC-3’) were ligated to the 5’ and 3’ cDNA ends. The cDNA carrying both, adapter A and adapter B attached to its ends was selected and then amplified with PCR using a proof reading enzyme (20 cycles) according to the 454 Life Science sequencing kit (454 Life Sciences, Branford, USA). Normalization was carried out by one cycle of denaturation and reassociation of the cDNA. Reassociated dscDNA was separated from the remaining ss-cDNA (normalized cDNA) by passing the mixture over a hydroxylapatite column. After hydroxylapatite chromatography, the sscDNA was amplified with 8 PCR cycles. Finally, the quality of this single-stranded template DNA (sstDNA) library was assessed using a 2100 BioAnalyzer (Agilent, Waldbronn, Germany). Approximately 10 μg of normalized double stranded cDNA was used for 454 pyrosequencing.    Pyrosequencing and de novo assembly  The four captured cDNA-sequencing libraries were prepared separately and each one was loaded on one of the four-lane gasket PicoTiterPlate devices (PTP; 70 × 75 mm; 454 Life Sciences, Branford, USA). Each library was quantitated by Pico-Green® (Quant-iT™, Molecular Probes, Invitrogen), diluted to 1 × 105 molecules/µL, and used for emPCR at a ratio of 0.1 copies of library fragments per DNA capture bead. The loaded PTP was then inserted into the GS-FLX Titanium instrument (Roche, Mannheim, Germany) for pyrosequencing, and sequencing reagents were sequentially flowed over the plate. Information from all the wells of the PTP was captured simultaneously by a camera and processed in real time by the onboard computer. The data from the 454 read sequences of each sample were assembled into contigs using the proprietary Roche 454 Newbler Assembler software (version 2.3; Roche). SolexaQA package software (version 1.13) was used to remove the low-quality sequences, end regions that were rich in ambiguous nucleotides, very short reads (< 50 bp), poly (A/T) tails, adaptors for cDNA synthesis, primers, and potentially contaminating vector sequences. After removing low-quality sequences and filtering out adaptors and primers, the curated raw 454 read sequences were assembled using the Roche 454 Newbler Assembler software.