Next Generation Sequencing
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  • 454™ Library Preparation

    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.
    • Tips for Optimizing DNA Inputs in NGS Library Construction

      Optimizing DNA inputs for NGS library prep is an important step, if you want to ensure a high-quality library. Start by following these tips.

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    • Why Choose NEBNext Reagents for NGS Library Prep?

      NEBNext products are a great choice for your NGS library preparation needs. In this video, Fiona shares a few reasons why.

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    • NEBNext for Core Lab Facilities

      Are you running a core sequencing facility? Watch as Eileen shares a few reasons that NEBNext products are particularly well suited to sequencing core labs.

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    • Tips for Optimizing RNA Inputs in NGS Library Construction

      Watch as Deyra explains how to optimize your RNA inputs for successful NGS library preparation.

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    • Which NEBNext Product Format Should I Choose?

      Not sure how sets and kits differ from modules? Ping explains the difference, and helps you to decide which one is right for you.

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    • 12 Quick Tips for NGS Library Preparation

      Using beads to clean up your DNA prior to NGS library prep can be quick and easy, if you follow these few, simple tips.

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    • Tips for Bead-based Clean ups and Size Selection

      Using beads to clean up your DNA prior to NGS library prep can be quick and easy, if you follow these few, simple tips.

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    Featured Products

    454™ Library Preparation includes these areas of focus:

    DNA for 454™
    RNA for 454™

    FAQs for 454™ Library Preparation

    Quick DNA Library Construction Workflow for 454

    DNA Library Construction Workflow for 454

    mRNA Library Construction Workflow for 454