Special features of RAD Sequencing data: implications for genotyping

Restriction site‐associated DNA Sequencing (RAD‐Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing‐by‐synthesis methods, RAD‐Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that t...

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Bibliographic Details
Published inMolecular ecology Vol. 22; no. 11; pp. 3151 - 3164
Main Authors Davey, John W., Cezard, Timothée, Fuentes-Utrilla, Pablo, Eland, Cathlene, Gharbi, Karim, Blaxter, Mark L.
Format Journal Article
LanguageEnglish
Published England Blackwell Publishing Ltd 01.06.2013
Subjects
Animals
Base Sequence
Caenorhabditis elegans - genetics
Chromosome Mapping - methods
contig assembly
Gene loci
Genetic Markers
Genotype
Genotype & phenotype
genotyping by sequencing
Genotyping Techniques
Heliconiaceae - genetics
High-Throughput Nucleotide Sequencing - methods
Polymorphism, Single Nucleotide
population genetics
RAD Sequencing
restriction enzymes
Restriction Mapping
Sequence Analysis, DNA
Software Pipelines
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Summary:Restriction site‐associated DNA Sequencing (RAD‐Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing‐by‐synthesis methods, RAD‐Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD‐Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD‐Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools.
Bibliography:ArticleID:MEC12084
Appendix S1 Additional methods Fig. S1 Effect of restriction fragment length in three RAD-Seq data sets. Fig. S2 Shearing of plasmids pOJ260 (3.5 kb, red) and pLUM (9 kb, blue). Fig. S3 Read depth varies with modified shearing conditions. Fig. S4 Heliconius allele counts cannot be separated by removing PCR duplicates. Fig. S5 Additional RAD contig assembly comparisons. Table S1 Caenorhabditis elegans PstI RAD library details. Table S2 Restriction site-associated DNA contig assembly details. Table S3 Restriction site-associated DNA adapter and primer sequences.
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ISSN:0962-1083
1365-294X
DOI:10.1111/mec.12084