Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD)

PeerJ

article

Natalia J. Bayona-Vásquez¹  Travis C. Glenn¹  Troy J. Kieran¹  Todd W. Pierson¹  Sandra L. Hoffberg⁴  Peter A. Scott⁸  Kerin E. Bentley⁴  John W. Finger¹  Swarnali Louha³  Nicholas Troendle⁴  Pindaro Diaz-Jaimes²  Rodney Mauricio⁴  Brant C. Faircloth^1,13 

[1] Department of Environmental Health Science, University of Georgia;Unidad Académica de Ecología y Biodiversidad Acuática, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México;Institute of Bioinformatics, University of Georgia;Department of Genetics, University of Georgia;Interdisciplinary Toxicology Program, University of Georgia;Department of Ecology and Evolutionary Biology, University of Tennessee;Department of Ecology, Evolution and Environmental Biology, Columbia University;Department of Biological Sciences, University of Alabama;Department of Ecology and Evolutionary Biology, University of California;LeafWorks Inc.;Department of Biological Sciences, Auburn University;Department of Natural, Health, and Mathematical Sciences, MidAmerica Nazarene University;Department of Biological Sciences and Museum of Natural Science, Louisiana State University

关键词: ddRAD;    Reduced representation library;    Restriction enzyme;    Next generation sequencing;    Illumina;    HiSeq;    NovaSeq;    Multiplexing;    In-line barcodes;    iTru;   

DOI  :  10.7717/peerj.7724

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

【 授权许可】

CC BY