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

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dc.contributor.authorBayona-Vasquez, Natalia J.
dc.contributor.authorGlenn, Travis C.
dc.contributor.authorKieran, Troy J.
dc.contributor.authorPierson, Todd W.
dc.contributor.authorHoffberg, Sandra L.
dc.contributor.authorScott, Peter A.
dc.contributor.authorBentley, Kerin E.
dc.contributor.authorFinger, John W.
dc.contributor.authorLouha, Swarnali
dc.contributor.authorTroendle, Nicholas
dc.contributor.authorDiaz-Jaimes, Pindaro
dc.contributor.authorMauricio, Rodney
dc.contributor.authorFaircloth, Brant C.
dc.contributor.otherUniversity of Georgia
dc.contributor.otherUniversidad Nacional Autonoma de Mexico
dc.contributor.otherUniversity of Alabama Tuscaloosa
dc.contributor.otherLouisiana State University
dc.contributor.otherUniversity of Tennessee Knoxville
dc.contributor.otherColumbia University
dc.contributor.otherUniversity of California Los Angeles
dc.contributor.otherAuburn University
dc.date.accessioned2023-10-02T15:16:05Z
dc.date.available2023-10-02T15:16:05Z
dc.date.issued2019
dc.description.abstractMolecular 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.en_US
dc.format.mediumelectronic
dc.format.mimetypeapplication/pdf
dc.identifier.citationBayona-Vásquez, N. J., Glenn, T. C., Kieran, T. J., Pierson, T. W., Hoffberg, S. L., Scott, P. A., Bentley, K. E., Finger, J. W., Louha, S., Troendle, N., Diaz-Jaimes, P., Mauricio, R., & Faircloth, B. C. (2019). Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD). In PeerJ (Vol. 7, p. e7724). PeerJ. https://doi.org/10.7717/peerj.7724
dc.identifier.doi10.7717/peerj.7724
dc.identifier.orcidhttps://orcid.org/0000-0002-3711-0727
dc.identifier.orcidhttps://orcid.org/0000-0003-0602-2289
dc.identifier.orcidhttps://orcid.org/0000-0001-7725-3637
dc.identifier.orcidhttps://orcid.org/0000-0003-0661-7821
dc.identifier.orcidhttps://orcid.org/0000-0002-3230-3538
dc.identifier.urihttps://ir.ua.edu/handle/123456789/12542
dc.languageEnglish
dc.language.isoen_US
dc.publisherPeerJ
dc.rights.licenseAttribution 4.0 International (CC BY 4.0)
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectddRAD
dc.subjectReduced representation library
dc.subjectRestriction enzyme
dc.subjectNext generation sequencing
dc.subjectIllumina
dc.subjectHiSeq
dc.subjectNovaSeq
dc.subjectMultiplexing
dc.subjectIn-line barcodes
dc.subjectiTru
dc.subjectCELLULAR-DNA CONTENT
dc.subjectGENOME-SIZE
dc.subjectSEQUENCE CAPTURE
dc.subjectFLOW-CYTOMETRY
dc.subjectINFERENCE
dc.subjectDISCOVERY
dc.subjectREMOVAL
dc.subjectSTACKS
dc.subjectDDRAD
dc.subjectTICK
dc.subjectMultidisciplinary Sciences
dc.titleAdapterama III: Quadruple-Indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD)en_US
dc.typeArticle
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