Next Generation Sequencing

Illumina® Library Preparation

Library preparation of fragmented DNA, ChIP-DNA, and cDNA prepared from mRNA, for use with the Illumina® sequencing platform, involves repair of 3’ and 5’ ends followed by the addition of a non-templated dA-tail before ligation to an adaptor, and PCR amplification. Small RNA libraries are constructed using a different workflow, in which adaptors are ligated directly to the small RNA molecules, followed by reverse transcription and PCR amplification. For more information, choose the workflow tabs below.

Libraries prepared in this manner are suitable for cluster generation, the next step in the Illumina sequencing workflow.

NEBNext® reagents are available for each step in the library preparation workflow for the Illumina platform. For DNA, ChIP, mRNA and small RNA library preparation for sequencing with the Illumina series of instruments, reagents from NEB are available in a set, master mix set or module format. Each of these reagents has been functionally validated by preparation of a genomic DNA, ChIP-Seq DNA, mRNA or small RNA library, followed by Illumina sequencing.

GenomeWeb article reviews NEBNext Small RNA Kits.

Illumina® is a registered trademark of Illumina, Inc.
NEBNext® is a registered trademark of New England Biolabs, Inc.
  1. 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.

  2. Tips for Optimizing RNA Inputs in NGS Library Construction

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

  3. 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.

  4. 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.

  5. 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.

  6. A Breakthrough Method of Small RNA Sample Preparation

    The common problem of adaptor dimer formation during small RNA library construction can be avoided by using NEBNext® protocols. Learn about this technique, and how it improves both performance and sensitivity in library construction.

  7. 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.

Featured Products

Illumina® Library Preparation includes these areas of focus:

NEBNext® Ultra™ II DNA Library Prep
NEBNext® Ultra™ II RNA Library Prep
DNA for Illumina®
RNA for Illumina®

FAQs for Illumina® Library Preparation

Protocols for Illumina® Library Preparation

    Other Tools & Resources


  • The NEBNext Sample Preparation Workflow Poster

    The NEBNext Sample Preparation Workflow Overviews & Products poster gives you at-a-glance workflow details for our DNA & RNA library prep products for Illumina & Ion Torrent, as well as our latest featured products.

Legal and Disclaimers

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

DNA Library Preparation Workflow for Illumina

RNA Library Preparation Workflow for Illumina

Small RNA Library Preparation Workflow for Illumina