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PeerJ. 2019 Oct 11;7:e7755. doi: 10.7717/peerj.7755. eCollection 2019.

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

Author information

1
Department of Environmental Health Science, University of Georgia, Athens, GA, United States of America.
2
Interdisciplinary Toxicology Program, University of Georgia, Athens, GA, United States of America.
3
Department of Genetics, University of Georgia, Athens, GA, United States of America.
4
Georgia Genomics and Bioinformatics Core, University of Georgia, Athens, GA, United States of America.
5
Institute of Bioinformatics, University of Georgia, Athens, GA, United States of America.
6
Current affiliation:  Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, United States of America.
7
Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA, United States of America.
8
Current affiliation:  Cornell Institute for Host-Microbe Interaction and Disease, Cornell University, Ithaca, United States of America.
9
Current affiliation:  Department of Biological Sciences, Auburn University, Auburn, AL, United States of America.
10
Current affiliation:  Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN, United States of America.
11
Current affiliation:  LeafWorks Inc., Sebastopol, CA, United States of America.
12
Current affiliation:  Department of Ecology, Evolution, and Environmental Biology, Columbia University, New York, NY, United States of America.
13
Laboratorio de Genética para la Conservación, Centro de Investigaciones Biológicas del Noroeste, SC, Instituto Politécnico Nacional, La Paz, Mexico.
14
Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California, Mexico.
15
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, United States of America.
16
Integrated Research and Development Laboratory, Marshfield Clinic Research Institute, Marshfield, WI, United States of America.
17
Department of Poultry Science, University of Georgia, Athens, GA, United States of America.
18
Department of Animal and Dairy Science, University of Georgia, Athens, GA, United States of America.
19
Department of Plant Biology, University of Georgia, Athens, GA, United States of America.
20
Department of Infectious Diseases, University of Georgia, Athens, GA, United States of America.
21
University of Alaska Museum, Fairbanks, AK, United States of America.
22
Department of Biological Sciences and Museum of Natural Science, Louisiana State University, Baton Rouge, LA, United States of America.

Abstract

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.

KEYWORDS:

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

Conflict of interest statement

Brant C. Faircloth is an Academic Editor for PeerJ. The EHS DNA lab (baddna.uga.edu) and Georgia Genomics Bioinformatics Core (dna.uga.edu) provide oligonucleotide aliquots and library preparation services at cost, including some oligonucleotides and services that make use of the adapters and primers presented in this manuscript.

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