Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)

PeerJ

article

Travis C. Glenn¹  Todd W. Pierson¹  Natalia J. Bayona-Vásquez¹  Troy J. Kieran¹  Sandra L. Hoffberg²  Jesse C. Thomas IV¹  Daniel E. Lefever⁸  John W. Finger¹  Bei Gao¹  Xiaoming Bian¹  Swarnali Louha⁴  Ramya T. Kolli³  Kerin E.Bentley²  Julie Rushmore^1,16  Kelvin Wong^1,18  Timothy I. Shaw⁴  Michael J. Rothrock Jr^2,21  Anna M. McKee^2,22  Tai L. Guo⁸  Rodney Mauricio²  MarirosaMolina^1,18  Brian S. Cummings³  Lawrence H. Lash^2,24  Kun Lu¹  Gregory S. Gilbert^2,26  Stephen P. Hubbell^2,27  Brant C. Faircloth^2,29 

[1]Department of Environmental Health Science, University of Georgia

[2]Department of Genetics, University of Georgia

[3]Interdisciplinary Toxicology Program, University of Georgia

[4]Institute of Bioinformatics, University of Georgia

[5]Department of Ecology and Evolutionary Biology, University of Tennessee

[6]Department of Ecology, Evolution, and Environmental Biology, Columbia University

[7]Division of STD Prevention, Centers for Disease Control and Prevention

[8]Department of Veterinary Biosciences and Diagnostic Imaging, University of Georgia

[9]Integrative Systems Biology and Drug Discovery Institute, University of Pittsburgh

[10]Department of Biological Sciences, Auburn University

[11]Department of Medicine, University of California

[12]Complex Carbohydrate Research Center and Department of Microbiology, University of Georgia

[13]Department of Pharmaceutical and Biomedical Sciences, University of Georgia

[14]Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park

[15]LeafWorks Inc.

[16]School of Ecology & College of Veterinary Medicine, University of Georgia

[17]Epicenter for Disease Dynamics, One Health Institute, School of Veterinary Medicine, University of California

[18]US Environmental Protection Agency

[19]California Water Service

[20]Department of Computational Biology, St. Jude Children’s Research Hospital

[21]U.S. National Poultry Research Center

[22]South Atlantic Water Science Center

[23]National Exposure Research Laboratory, US Environmental Protection Agency, Research Triangle Park

[24]Department of Pharmacology, Wayne State University

[25]Department of Environmental Sciences and Engineering, University of North Carolina

[26]Environmental Studies Department, University of California

[27]Smithsonian Tropical Research Institute

[28]Department of Ecology and Evolutionary Biology, University of California

[29]Department of Biological Sciences and Museum of Natural Science, Louisiana State University

关键词: MiSeq;    Next generation sequencing;    Quadruple indexing;    Hierarchical indexing;    Multiplexing;    Fusion primers;    Internal tagging;    PCR;    Libraries;   

DOI  :  10.7717/peerj.7786

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

【 授权许可】

CC BY