Illumina® Library Preparation

Library preparation for the Illumina® sequencing platform requires inputs of a defined length, therefore fragmentation of DNA or the use of cDNA prepared from RNA is the starting point. This is followed by end repair (3’ and 5’) to generate blunt-ended, phosphorylated molecules, followed by 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, methylated DNA, microbiome DNA, total RNA, single cell/low input RNA, and small-RNA samples. These are complemented by a wide range of index primers for multiplexing. 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.


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:
NEBNext® Ultra™ II RNA Library Prep
NEBNext® Ultra™ II DNA Library Prep
RNA for Illumina®
DNA for Illumina®
FAQs for Illumina® Library Preparation
Protocols for Illumina® Library Preparation
Application Notes 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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