Crab-eating monkey
The first step of the preparation is the fragmentation of the high molecular weight DNA sample into smaller molecular species appropriate for sequencing using the GS FLX Titanium chemistry. This is done by nebulization, which shears double-stranded DNA into fragments ranging from about 400 to 1000 base pairs. This population of smaller-sized DNA species, generated from a single DNA sample, is referred to as a ???library???. 3~5ug of the cDNA was used to generate DNA library for Genome Sequencer FLX Titanium (Roche, Mannheim, GE). The fragments ends were polished (blunted), and short two adapters were ligated onto both ends. The adapters provide priming sequences for both amplification and sequencing of the sample library fragments, as well as the ???sequencing key???, a short sequence of 4 nucleotides used by the system software for base calling and, following repair of any nicks in the double-stranded library, release of the unbound strand of each fragment(with 5,-Adaptor A). Finally the quality of the library of single-stranded template DNA fragments(sst DNA library) was assessed using 2100 BioAnalyzer (Agilent, Waldbronn, GE), and the library was quantitated, including a functional quantitation to determine the optimal amount of the library to use as input for emulsion-based clonal amplification.
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