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

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.

[1]  Cui Jianxun,et al.  Nuclear DNA Content Variation in Fishes , 1991 .

[2]  R. Hinegardner,et al.  The cellular DNA content of sharks, rays and some other fishes. , 1976, Comparative biochemistry and physiology. B, Comparative biochemistry.

[3]  Deren A. R. Eaton,et al.  PyRAD: assembly of de novo RADseq loci for phylogenetic analyses , 2013, bioRxiv.

[4]  T R Tiersch,et al.  Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. , 1989, Cytometry.

[5]  Robert J. Elshire,et al.  A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species , 2011, PloS one.

[6]  T. Glenn,et al.  Isolating microsatellite DNA loci. , 2005, Methods in enzymology.

[7]  Travis C Glenn,et al.  RADcap: sequence capture of dual‐digest RADseq libraries with identifiable duplicates and reduced missing data , 2016, Molecular ecology resources.

[8]  B. Faircloth,et al.  Capturing Darwin's dream , 2016, Molecular ecology resources.

[9]  A. Amores,et al.  Stacks: Building and Genotyping Loci De Novo From Short-Read Sequences , 2011, G3: Genes | Genomes | Genetics.

[10]  P. Donnelly,et al.  Inference of population structure using multilocus genotype data. , 2000, Genetics.

[11]  Swarnali Louha,et al.  A High-Quality Reference Genome for the Invasive Mosquitofish Gambusia affinis Using a Chicago Library , 2018, G3: Genes, Genomes, Genetics.

[12]  C. Hill,et al.  Variation in genome size of argasid and ixodid ticks. , 2007, Insect biochemistry and molecular biology.

[13]  Jessica D. Stephens,et al.  Targeted DNA Region Re-sequencing , 2016 .

[14]  Cameron S. Osborne,et al.  Large Scale Loss of Data in Low-Diversity Illumina Sequencing Libraries Can Be Recovered by Deferred Cluster Calling , 2011, PloS one.

[15]  Troy J. Kieran,et al.  Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq) , 2015, Molecular ecology resources.

[16]  Trevor W. Rife,et al.  Genotyping‐by‐Sequencing for Plant Breeding and Genetics , 2012 .

[17]  M. Blaxter,et al.  Genome-wide genetic marker discovery and genotyping using next-generation sequencing , 2011, Nature Reviews Genetics.

[18]  E. Pante,et al.  Use of RAD sequencing for delimiting species , 2014, Heredity.

[19]  P. Etter,et al.  Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers , 2008, PloS one.

[20]  Konrad Bachmann,et al.  Feulgen slope determinations of urodele nuclear DNA amounts , 2004, Histochemie.

[21]  G. Luikart,et al.  Harnessing the power of RADseq for ecological and evolutionary genomics , 2016, Nature Reviews Genetics.

[22]  Aaron B. A. Shafer,et al.  Bioinformatic processing of RAD‐seq data dramatically impacts downstream population genetic inference , 2017 .

[23]  L. Lowcock,et al.  Genome size and metabolic rate in salamanders , 1991 .

[24]  Christopher E. Bird,et al.  ezRAD: a simplified method for genomic genotyping in non-model organisms , 2013, PeerJ.

[25]  R. Oono,et al.  Double‐digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles , 2018, Ecology and evolution.

[26]  E. Olmo,et al.  Genome size in some reptiles , 1976 .

[27]  J. Wiens,et al.  How Should Genes and Taxa be Sampled for Phylogenomic Analyses with Missing Data? An Empirical Study in Iguanian Lizards. , 2016, Systematic biology.

[28]  Florian Leese,et al.  Detection and Removal of PCR Duplicates in Population Genomic ddRAD Studies by Addition of a Degenerate Base Region (DBR) in Sequencing Adapters , 2014, The Biological Bulletin.

[29]  J. DeWoody,et al.  On the estimation of genome-wide heterozygosity using molecular markers. , 2005, The Journal of heredity.

[30]  B. Faircloth,et al.  Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels , 2012, PloS one.

[31]  M. Rowicka,et al.  Strategies for Achieving High Sequencing Accuracy for Low Diversity Samples and Avoiding Sample Bleeding Using Illumina Platform , 2015, PloS one.

[32]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[33]  M. Schartl,et al.  Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: detection of triploid Poecilia formosa. , 2000, Cytometry.

[34]  Gordon Luikart,et al.  Trade‐offs and utility of alternative RADseq methods: Reply to Puritz et al. , 2014, Molecular ecology.

[35]  Angel Amores,et al.  Stacks: an analysis tool set for population genomics , 2013, Molecular ecology.

[36]  Paul D N Hebert,et al.  The nucleotypic effects of cellular DNA content in cartilaginous and ray-finned fishes. , 2003, Genome.

[37]  A. Amores,et al.  Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers. , 2007, Genome research.

[38]  Travis C Glenn,et al.  Avoiding Missing Data Biases in Phylogenomic Inference: An Empirical Study in the Landfowl (Aves: Galliformes). , 2016, Molecular biology and evolution.

[39]  H. Hoekstra,et al.  Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species , 2012, PloS one.

[40]  L. Duret,et al.  Is RAD-seq suitable for phylogenetic inference? An in silico assessment and optimization , 2013, Ecology and evolution.

[41]  L. Rocco,et al.  Genome size and A-T rich DNA in selachians , 1989, Genetica.

[42]  E. Olmo,et al.  Further Data on the Genome Size in the Urodeles , 1974 .

[43]  Axel Meyer,et al.  quaddRAD: a new high‐multiplexing and PCR duplicate removal ddRAD protocol produces novel evolutionary insights in a nonradiating cichlid lineage , 2017, Molecular ecology.

[44]  Travis C Glenn,et al.  Sequence Capture versus Restriction Site Associated DNA Sequencing for Shallow Systematics. , 2013, Systematic biology.

[45]  Omar Triana,et al.  Morphometric and molecular evidence of intraspecific biogeographical differentiation of Rhodnius pallescens (HEMIPTERA: REDUVIIDAE: RHODNIINI) from Colombia and Panama. , 2012, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[46]  J. Bantle,et al.  Genome size and organization in the ixodid tick Amblyomma americanum (L.) , 1994, Insect molecular biology.

[47]  Travis C Glenn,et al.  Resolving taxonomic turbulence and uncovering cryptic diversity in the musk turtles (Sternotherus) using robust demographic modeling. , 2018, Molecular phylogenetics and evolution.

[48]  M. Matz,et al.  2b-RAD: a simple and flexible method for genome-wide genotyping , 2012, Nature Methods.

[49]  Romdhane Rekaya,et al.  Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) , 2019, PeerJ.

[50]  F. Guerrero,et al.  Genome size and organization in the blacklegged tick, Ixodes scapularis and the Southern cattle tick, Boophilus microplus , 2005, Insect molecular biology.

[51]  P. Hebert,et al.  Genome-size evolution in fishes , 2004 .

[52]  Y. Ojima,et al.  Cellular DNA contents of fishes determined by flow cytometry , 1990 .