Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq)

Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next‐generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double‐digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0‐, 12‐, 48‐ and 96‐h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12–48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96‐h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2‐ to 16‐fold), and additional read‐length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

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

[2]  B. Deagle,et al.  Fine‐scale diet of the Australian sea lion (Neophoca cinerea) using DNA‐based analysis of faeces , 2015 .

[3]  G. Vendramin,et al.  Genomic exploration and molecular marker development in a large and complex conifer genome using RADseq and mRNAseq , 2015, Molecular ecology resources.

[4]  B. Emerson,et al.  Restriction site‐associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference , 2015, Molecular ecology resources.

[5]  Á. Carracedo,et al.  A SNaPshot of next generation sequencing for forensic SNP analysis. , 2015, Forensic science international. Genetics.

[6]  C. Somers,et al.  Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings. , 2015, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[7]  H. Rezaei,et al.  Is black coat color in wolves of Iran an evidence of admixed ancestry with dogs? , 2015, Journal of Applied Genetics.

[8]  D. Bolnick,et al.  Demystifying the RAD fad , 2014, Molecular ecology.

[9]  H. Kühl,et al.  The genetic population structure of wild western lowland gorillas (Gorilla gorilla gorilla) living in continuous rain forest , 2014, American journal of primatology.

[10]  S. Lien,et al.  Genome‐wide SNP analysis reveals a genetic basis for sea‐age variation in a wild population of Atlantic salmon (Salmo salar) , 2014, Molecular ecology.

[11]  S. Long,et al.  A footprint of past climate change on the diversity and population structure of Miscanthus sinensis. , 2014, Annals of botany.

[12]  Joanna Y. Wilson,et al.  A Self‐Contained, Controlled Hatchery System for Rearing Lake Whitefish Embryos for Experimental Aquaculture , 2014 .

[13]  A. Mikheyev,et al.  Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics , 2014, PloS one.

[14]  T. Jombart,et al.  How reliable are morphological and anatomical characters to distinguish European wildcats, domestic cats and their hybrids in France? , 2014 .

[15]  Jean-Marie Rouillard,et al.  Ancient whole genome enrichment using baits built from modern DNA. , 2014, Molecular biology and evolution.

[16]  Nathaniel D. Chu,et al.  Phylogenomic analyses reveal latitudinal population structure and polymorphisms in heat stress genes in the North Atlantic snail Nucella lapillus , 2014, Molecular ecology.

[17]  Jeffrey D. Lozier,et al.  Revisiting comparisons of genetic diversity in stable and declining species: assessing genome‐wide polymorphism in North American bumble bees using RAD sequencing , 2014, Molecular ecology.

[18]  Shizhong Xu,et al.  Population genomic analyses from low-coverage RAD-Seq data: a case study on the non-model cucurbit bottle gourd. , 2014, The Plant journal : for cell and molecular biology.

[19]  L. Seeb,et al.  Genotyping by sequencing resolves shallow population structure to inform conservation of Chinook salmon (Oncorhynchus tshawytscha) , 2014, Evolutionary applications.

[20]  N. Barton,et al.  Likelihood‐based inference of population history from low‐coverage de novo genome assemblies , 2014, Molecular ecology.

[21]  B. Llamas,et al.  DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification , 2013, Investigative Genetics.

[22]  J. Slate,et al.  FINE‐SCALE GENETIC STRUCTURE IN A WILD BIRD POPULATION: THE ROLE OF LIMITED DISPERSAL AND ENVIRONMENTALLY BASED SELECTION AS CAUSAL FACTORS , 2013, Evolution; international journal of organic evolution.

[23]  N. Street,et al.  De Novo SNP Discovery in the Scandinavian Brown Bear (Ursus arctos) , 2013, PloS one.

[24]  Martin Sikora,et al.  Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries. , 2013, American journal of human genetics.

[25]  L. Bernatchez,et al.  Targeted sequence capture and resequencing implies a predominant role of regulatory regions in the divergence of a sympatric lake whitefish species pair (Coregonus clupeaformis) , 2013, Molecular ecology.

[26]  L. Bernatchez,et al.  THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION DURING SPECIATION‐WITH‐GENE‐FLOW IN LAKE WHITEFISH SPECIES PAIRS ASSESSED BY RAD SEQUENCING , 2013, Evolution; international journal of organic evolution.

[27]  B. Guldbrandtsen,et al.  The admixed population structure in Danish Jersey dairy cattle challenges accurate genomic predictions. , 2013, Journal of animal science.

[28]  K. Gharbi,et al.  Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing , 2013, Molecular ecology.

[29]  G. Luikart,et al.  Genomic patterns of introgression in rainbow and westslope cutthroat trout illuminated by overlapping paired‐end RAD sequencing , 2013, Molecular ecology.

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

[31]  R. Crawford,et al.  Genome sequence of dwarf birch (Betula nana) and cross‐species RAD markers , 2013, Molecular ecology.

[32]  Z. Iqbal,et al.  Reference‐free SNP discovery for the Eurasian beaver from restriction site–associated DNA paired‐end data , 2013, Molecular ecology.

[33]  S. Narum,et al.  Genotyping‐by‐sequencing in ecological and conservation genomics , 2013, Molecular ecology.

[34]  Heebal Kim,et al.  Genetic diversity, population structure and relationships in indigenous cattle populations of Ethiopia and Korean Hanwoo breeds using SNP markers , 2013, Front. Genet..

[35]  Wendy S. Schackwitz,et al.  A 34K SNP genotyping array for Populus trichocarpa: Design, application to the study of natural populations and transferability to other Populus species , 2013, Molecular ecology resources.

[36]  J. Reif,et al.  Population structure, genetic diversity and linkage disequilibrium in elite winter wheat assessed with SNP and SSR markers , 2013, Theoretical and Applied Genetics.

[37]  Lusheng Huang,et al.  Genetic Diversity, Linkage Disequilibrium and Selection Signatures in Chinese and Western Pigs Revealed by Genome-Wide SNP Markers , 2013, PloS one.

[38]  M. Groenen,et al.  Genome‐wide single nucleotide polymorphism analysis reveals recent genetic introgression from domestic pigs into Northwest European wild boar populations , 2013, Molecular ecology.

[39]  L. Bernatchez,et al.  SNP‐array reveals genome‐wide patterns of geographical and potential adaptive divergence across the natural range of Atlantic salmon (Salmo salar) , 2013, Molecular ecology.

[40]  Brian Boyle,et al.  An Improved Genotyping by Sequencing (GBS) Approach Offering Increased Versatility and Efficiency of SNP Discovery and Genotyping , 2013, PloS one.

[41]  T. Cezard,et al.  Special features of RAD Sequencing data: implications for genotyping , 2012, Molecular ecology.

[42]  Nnamdi E. Ihuegbu,et al.  Microhaplotype loci are a powerful new type of forensic marker , 2013 .

[43]  A. LarsonWesley,et al.  Single-nucleotide polymorphisms reveal distribution and migration of Chinook salmon (Oncorhynchus tshawytscha) in the Bering Sea and North Pacific Ocean , 2013 .

[44]  Molecular forensics in the precious Mediterranean red coral, Corallium rubrum: testing DNA extraction and microsatellite genotyping using dried colonies , 2013, Conservation Genetics Resources.

[45]  Matthew Mayho,et al.  Evaluation and optimisation of preparative semi‐automated electrophoresis systems for Illumina library preparation , 2012, Electrophoresis.

[46]  K. Glover,et al.  Three Decades of Farmed Escapees in the Wild: A Spatio-Temporal Analysis of Atlantic Salmon Population Genetic Structure throughout Norway , 2012, PloS one.

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

[48]  J. Obeso,et al.  Population genetic structure and diversity of the endangered Cantabrian capercaillie , 2012 .

[49]  L. Orlando,et al.  Next-generation sequencing offers new insights into DNA degradation. , 2012, Trends in biotechnology.

[50]  J. Birks,et al.  Molecular comparison of historical and contemporary pine marten (Martes martes) populations in the British Isles: evidence of differing origins and fates, and implications for conservation management , 2012, Conservation Genetics.

[51]  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.

[52]  Y. Kaneko,et al.  Genetic population structure of the masked palm civet Paguma larvata, (Carnivora: Viverridae) in Japan, revealed from analysis of newly identified compound microsatellites , 2012, Conservation Genetics.

[53]  Resurrecting an extinct salmon evolutionarily significant unit: archived scales, historical DNA and implications for restoration , 2012, Molecular ecology.

[54]  J. Poland,et al.  Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach , 2012, PloS one.

[55]  N. Kyrpides,et al.  Direct Comparisons of Illumina vs. Roche 454 Sequencing Technologies on the Same Microbial Community DNA Sample , 2012, PloS one.

[56]  C. Doe,et al.  A conserved haplotype controls parallel adaptation in geographically distant salmonid populations , 2012, Molecular ecology.

[57]  N. Wagemaker,et al.  Genomic toolboxes for conservation biologists , 2011, Evolutionary applications.

[58]  Juliane C. Dohm,et al.  Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems , 2011, Genome Biology.

[59]  T. Glenn Field guide to next‐generation DNA sequencers , 2011, Molecular ecology resources.

[60]  Detlef Weigel,et al.  Paired-end RAD-seq for de novo assembly and marker design without available reference , 2011, Bioinform..

[61]  J. Galindo,et al.  Applications of next generation sequencing in molecular ecology of non-model organisms , 2011, Heredity.

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

[63]  L. Seeb,et al.  Single-Nucleotide Polymorphisms (SNPs) under Diversifying Selection Provide Increased Accuracy and Precision in Mixed-Stock Analyses of Sockeye Salmon from the Copper River, Alaska , 2011 .

[64]  G. Valè,et al.  Identification of SNP and SSR markers in eggplant using RAD tag sequencing , 2011, BMC Genomics.

[65]  S. Salvi,et al.  High-throughput SNP discovery and genotyping in durum wheat (Triticum durum Desf.) , 2011, Theoretical and Applied Genetics.

[66]  Margaret C. Linak,et al.  Sequence-specific error profile of Illumina sequencers , 2011, Nucleic acids research.

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

[68]  T. Burke,et al.  DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species , 2011, Molecular ecology resources.

[69]  Rebekah A. Oomen,et al.  Panmixia on a continental scale in a widely distributed colonial waterbird , 2011 .

[70]  Eric A. Johnson,et al.  Mapping with RAD (restriction-site associated DNA) markers to rapidly identify QTL for stem rust resistance in Lolium perenne , 2011, Theoretical and Applied Genetics.

[71]  L. Bernatchez,et al.  SNP signatures of selection on standing genetic variation and their association with adaptive phenotypes along gradients of ecological speciation in lake whitefish species pairs (Coregonus spp.) , 2011, Molecular ecology.

[72]  Edward S. Buckler,et al.  Genetic structure and domestication history of the grape , 2011, Proceedings of the National Academy of Sciences.

[73]  Christophe Dessimoz,et al.  Base-calling for next-generation sequencing platforms , 2011, Briefings Bioinform..

[74]  I. Jamieson,et al.  Historic DNA reveals contemporary population structure results from anthropogenic effects, not pre-fragmentation patterns , 2011, Conservation Genetics.

[75]  John C. Carlson,et al.  Population structure and genetic diversity of greater sage-grouse (Centrocercus urophasianus) in fragmented landscapes at the northern edge of their range , 2011, Conservation Genetics.

[76]  M. Hofreiter,et al.  Next Generation Sequencing of Ancient DNA: Requirements, Strategies and Perspectives , 2010, Genes.

[77]  D. Neale,et al.  Patterns of Population Structure and Environmental Associations to Aridity Across the Range of Loblolly Pine (Pinus taeda L., Pinaceae) , 2010, Genetics.

[78]  L. Bernatchez,et al.  On the origin of species: insights from the ecological genomics of lake whitefish , 2010, Philosophical Transactions of the Royal Society B: Biological Sciences.

[79]  K. Røed,et al.  Museum specimens reveal changes in the population structure of northern Fennoscandian domestic reindeer in the past one hundred years. , 2010, Animal genetics.

[80]  M. Metzker Sequencing technologies — the next generation , 2010, Nature Reviews Genetics.

[81]  T. Als,et al.  A comparison of SNP and STR loci for delineating population structure and performing individual genetic assignment , 2010, BMC Genetics.

[82]  O. Seehausen,et al.  Divergence along a steep ecological gradient in lake whitefish (Coregonus sp.) , 2009, Journal of evolutionary biology.

[83]  S. Narum,et al.  Quantitative PCR assessment of microsatellite and SNP genotyping with variable quality DNA extracts , 2009, Conservation Genetics.

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

[85]  Juliane C. Dohm,et al.  Substantial biases in ultra-short read data sets from high-throughput DNA sequencing , 2008, Nucleic acids research.

[86]  C. Millar,et al.  New developments in ancient genomics. , 2008, Trends in ecology & evolution.

[87]  Robert D Schnabel,et al.  SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries , 2008, Nature Methods.

[88]  E. Mardis The impact of next-generation sequencing technology on genetics. , 2008, Trends in genetics : TIG.

[89]  Jan van Oeveren,et al.  Complexity Reduction of Polymorphic Sequences (CRoPS™): A Novel Approach for Large-Scale Polymorphism Discovery in Complex Genomes , 2007, PloS one.

[90]  Philip L. F. Johnson,et al.  Patterns of damage in genomic DNA sequences from a Neandertal , 2007, Proceedings of the National Academy of Sciences.

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

[92]  W Parson,et al.  Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise. , 2006, Forensic science international.

[93]  C. P. Madenjian,et al.  Energy Density of Lake Whitefish Coregonus clupeaformis in Lakes Huron and Michigan , 2006, Environmental Biology of Fishes.

[94]  E. Anderson,et al.  The Power of Single-Nucleotide Polymorphisms for Large-Scale Parentage Inference , 2006, Genetics.

[95]  M. Apollonio,et al.  An empirical approach for reliable microsatellite genotyping of wolf DNA from multiple noninvasive sources , 2006, Conservation Genetics.

[96]  J. S. Quinn,et al.  Non-destructive sampling of maternal DNA from the external shell of bird eggs , 2006, Conservation Genetics.

[97]  C. Krebs,et al.  Monitoring coyote population dynamics by genotyping faeces , 2005, Molecular ecology.

[98]  L. Bernatchez,et al.  FAST‐TRACK: Integrating QTL mapping and genome scans towards the characterization of candidate loci under parallel selection in the lake whitefish (Coregonus clupeaformis) , 2004, Molecular ecology.

[99]  P. Taberlet,et al.  How to track and assess genotyping errors in population genetics studies , 2004, Molecular ecology.