Two methods are available for library preparation of fragmented DNA for the 454™ sequencing platform. The “quick” library preparation method includes repair of DNA ends, and the addition of a non-templated dAMP. Libraries are precipitated to select appropriately sized fragments before ligation to an adaptor. Libraries are prepared from mRNA by construction of a cDNA library followed by preparation of a DNA library using the quick library preparation method (see tab below). The “quick” library preparation method includes repair of DNA ends, and the addition of a non-templated dA-tail, followed by ligation to an adaptor. The original method for fragmented DNA preparation is no longer commonly used. This protocol includes repair of DNA ends, before ligation to an adaptor. Hydrophilic streptavidin magnetic beads are added to library to biotinylate one adaptor per fragment. Upon ligation of the fragment and the unphosphorylated adaptor, a nick in the adaptor sequence is generated. The nicked strand is displaced, the 3’ end of the DNA template is extended, and the adaptor sequence is filled-in to generate full-length dsDNA. This process is coupled with the elution of the full-length, unbiotinylated ssDNA strand. For more information, choose the workflow tabs below.
Libraries prepared by these methods are suitable for capture bead immobilization and emulsion PCR, the next step in the 454 sequencing workflow.
NEBNext® reagents are available for each step in the DNA library preparation workflow for the 454 platform. For DNA or mRNA, reagents from NEB are available in a set, master mix or module format. Each reagent has been functionally validated by preparation of a genomic DNA library or RNA library from a standard reference followed by 454 sequencing.
454™ is a trademark of Roche.
NEBNext® is a registered trademark of New England Biolabs, Inc.