Illumina® Library Preparation

Library preparation for the Illumina sequencing platform requires fragmentation of DNA, or use of cDNA prepared from RNA, followed by repair of 3’ and 5’ ends to form blunt-ended, phosphorylated molecules, and the addition of a non-templated dA-tail before ligation to an adaptor. If necessary to achieve sufficient yields, the final step is PCR amplification of the library. 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 detail, select a workflow tab below.

The NEBNext suite of products supports Illumina sequencing with sample preparation tools that streamline workflows, minimize inputs, and improve library yields and quality. NEBNext sample preparation kits are available for genomic DNA, ChIP DNA, FFPE DNA, Microbiome DNA, RNA and small RNA samples. In addition to the extensive QCs performed on individual kit components, all NEBNext kits for Illumina are functionally validated by preparation of a library, followed by Illumina sequencing. NEBNext library preparation reagents are available in multiple kit and workflow formats, for maximum convenience and flexibility.

GenomeWeb article reviews NEBNext Small RNA Kits.

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

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Illumina® Library Preparation includes these areas of focus:

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

FAQs for Illumina® Library Preparation

Protocols for Illumina® Library Preparation

Single Cell/Low Input RNA Library Preparation Workflow for Illumina


Ultra II FS DNA Library Preparation Workflow


Ultra II Directional RNA Library Preparation Workflow for Illumina


Ultra II  Non-directional RNA Library Preparation Workflow for Illumina


Ultra II DNA Library Preparation Workflow for Illumina

Ultra DNA Library Preparation Workflow for Illumina

Small RNA Library Preparation Workflow for Illumina


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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.