高通量测序Meta 16S建库引物设计-双barcode

高通量测序Meta 16S建库引物设计-双barcode

高通量测序Meta 16S建库引物设计-双barcode

An improved dual-indexing approach for

multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform

Douglas W Fadrosh 1†,Bing Ma 1†,Pawel Gajer 1,Naomi Sengamalay 1,Sandra Ott 1,Rebecca M Brotman 2and Jacques Ravel 1*

Background

The development of methods to detect fastidious or non-cultivable organisms through amplification and determination of the sequence of conserved genes,or culture-independent profiling,has precipitated a revo-lution in biology.It was recognized decades ago that the number of microbes seen on direct staining of environmental or human samples often exceeded by many orders of magnitude the number that could be cultured (termed "the great plate-count anomaly")[1].Culture-independent profiling of bacterial communities relies on the amplification and sequencing of the generally considered universal 16S rRNA gene and has greatly increased appreciation for the complexity hidden in even seemingly simple microbial consortia.Advance-ments in next-generation sequencing technologies,in

terms of throughput,sequence read length and accur-acy,has had a major impact in the field by enabling large numbers of samples to be examined at greater depth.The Illumina MiSeq platform (San Diego,CA,USA)provides researchers with a scalable,high-throughput and streamlined sequencing platform to survey com-munity composition from clinical and environmental samples.However,known limitations with the MiSeq platform associated with the sequencing of low sequence diversity samples has hampered harnessing its true poten-tial to sequence 16S rRNA gene amplicons.The “low sequence diversity ”issue arises in the first several cycles of a Miseq 16S rRNA gene amplicon sequencing run,during which successful cluster identification and phasing/pre-phasing calibration are dependent on heterogeneous base composition of targeted amplicons.Because of the nature of the 16S rRNA gene,amplicon pools are highly homogenous and are required to be co-sequenced with a heterogeneous control library,commonly phage PhiX,normally combined 1:1with the amplicon pool.This improves the quality of the sequencing reads enough to yield a successful sequencing run (average quality value

*Correspondence:jravel@som.umaryland.edu †

Equal contributors 1

Institute for Genome Sciences,Department of Microbiology and

Immunology,University of Maryland School of Medicine,801W.Baltimore Street,Baltimore,MD 21201,USA

Full list of author information is available at the end of the

高通量测序Meta 16S建库引物设计-双barcode

高通量测序Meta 16S建库引物设计-双barcode

article

©2014Fadrosh et al.;licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://m.wendangku.net/doc/26b0a9cf960590c69fc3760a.html /licenses/by/2.0),which permits unrestricted use,distribution,and reproduction in any medium,provided the original work is properly credited.The Creative Commons Public Domain Dedication waiver (http://m.wendangku.net/doc/26b0a9cf960590c69fc3760a.html /publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.

Fadrosh et al.Microbiome 2014,2:6

http://m.wendangku.net/doc/26b0a9cf960590c69fc3760a.html /content/2/1/6

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