Open Access Highly Accessed Methodology article

Multiplexed Illumina sequencing libraries from picogram quantities of DNA

Sarah K Bowman1*, Matthew D Simon14, Aimee M Deaton1, Michael Tolstorukov235, Mark L Borowsky16 and Robert E Kingston1

Author Affiliations

1 Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02114, USA

2 Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA

3 Division of Genetics, Brigham and Women’s Hospital, Boston, MA 02115, USA

4 Present address: Department of Molecular Biophysics and Biochemistry and Chemical Biology Institute, Yale University, West Haven, CT 06516, USA

5 Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA

6 Present address: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA

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BMC Genomics 2013, 14:466  doi:10.1186/1471-2164-14-466

Published: 9 July 2013



High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps.


We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment.


Application of this method allows whole genome studies from samples where material or yields are limiting.

Illumina; ChIP-seq; Multiplex; Barcoding; Library preparation