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Nucleic Acids Research Advance Access published online on October 11, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm760
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing

Poornima Parameswaran1, Roxana Jalili3, Li Tao4, Shadi Shokralla3, Baback Gharizadeh3, Mostafa Ronaghi3 and Andrew Z. Fire2,*

1Department of Microbiology and Immunology, 2Department of Pathology and Department of Genetics, Stanford University School of Medicine, 3Stanford Genome Technology Center and 4Department of Biological Sciences, Stanford University, Stanford, CA-94305, USA

* To whom correspondence should be addressed. Tel: +1 650 723 2885; Fax: +1 650 724 9070; Email: afire{at}stanford.edu

Received July 30, 2007. Revised September 10, 2007. Accepted September 11, 2007.

Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward–reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n2 independent libraries for each set of ‘n’ forward and ‘n’ reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.


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