Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)

PeerJ

article

Travis C. Glenn¹  Roger A. Nilsen⁴  Troy J. Kieran¹  Jon G. Sanders⁷  Natalia J. Bayona-Vásquez¹  John W. Finger¹  Todd W. Pierson¹  Kerin E. Bentley³  Sandra L. Hoffberg³  Swarnali Louha⁵  Francisco J. Garcia-De Leon^1,13  Miguel Angel del Rio Portilla^1,14  Kurt D. Reed^1,15  Jennifer L. Anderson^1,16  Jennifer K. Meece^1,16  Samuel E. Aggrey⁵  Romdhane Rekaya⁵  Magdy Alabady⁴  Myriam Belanger⁴  Kevin Winker^2,21  Brant C. Faircloth^2,22 

[1] Department of Environmental Health Science, University of Georgia;Interdisciplinary Toxicology Program, University of Georgia;Department of Genetics, University of Georgia;Georgia Genomics and Bioinformatics Core, University of Georgia;Institute of Bioinformatics, University of Georgia;Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia;Department of Pediatrics, School of Medicine, University of California San Diego;Cornell Institute for Host—Microbe Interaction and Disease, Cornell University;Department of Biological Sciences, Auburn University;Department of Ecology and Evolutionary Biology, University of Tennessee;LeafWorks Inc.;Department of Ecology, Evolution, and Environmental Biology, Columbia University;Laboratorio de Genética para la Conservación, Centro de Investigaciones Biológicas del Noroeste, SC, Instituto Politécnico Nacional;Centro de Investigación Científica y de Educación Superior de Ensenada;Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison;Integrated Research and Development Laboratory, Marshfield Clinic Research Institute;Department of Poultry Science, University of Georgia;Department of Animal and Dairy Science, University of Georgia;Department of Plant Biology, University of Georgia;Department of Infectious Diseases, University of Georgia;University of Alaska Museum;Department of Biological Sciences and Museum of Natural Science, Louisiana State University

关键词: Illumina;    Next Generation Sequencing;    NovaSeq;    Sample Preparation;    Pooling;    Multiplexing;    Adapters;    Primers;   

DOI  :  10.7717/peerj.7755

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

【 授权许可】

CC BY