Dataset Information

Amplification biases and consistent recovery of loci in a double-digest RAD-seq protocol.

ABSTRACT: A growing variety of "genotype-by-sequencing" (GBS) methods use restriction enzymes and high throughput DNA sequencing to generate data for a subset of genomic loci, allowing the simultaneous discovery and genotyping of thousands of polymorphisms in a set of multiplexed samples. We evaluated a "double-digest" restriction-site associated DNA sequencing (ddRAD-seq) protocol by 1) comparing results for a zebra finch (Taeniopygia guttata) sample with in silico predictions from the zebra finch reference genome; 2) assessing data quality for a population sample of indigobirds (Vidua spp.); and 3) testing for consistent recovery of loci across multiple samples and sequencing runs. Comparison with in silico predictions revealed that 1) over 90% of predicted, single-copy loci in our targeted size range (178-328 bp) were recovered; 2) short restriction fragments (38-178 bp) were carried through the size selection step and sequenced at appreciable depth, generating unexpected but nonetheless useful data; 3) amplification bias favored shorter, GC-rich fragments, contributing to among locus variation in sequencing depth that was strongly correlated across samples; 4) our use of restriction enzymes with a GC-rich recognition sequence resulted in an up to four-fold overrepresentation of GC-rich portions of the genome; and 5) star activity (i.e., non-specific cutting) resulted in thousands of "extra" loci sequenced at low depth. Results for three species of indigobirds show that a common set of thousands of loci can be consistently recovered across both individual samples and sequencing runs. In a run with 46 samples, we genotyped 5,996 loci in all individuals and 9,833 loci in 42 or more individuals, resulting in <1% missing data for the larger data set. We compare our approach to similar methods and discuss the range of factors (fragment library preparation, natural genetic variation, bioinformatics) influencing the recovery of a consistent set of loci among samples.

SUBMITTER: DaCosta JM

PROVIDER: S-EPMC4154734 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Amplification biases and consistent recovery of loci in a double-digest RAD-seq protocol.

DaCosta Jeffrey M JM Sorenson Michael D MD

PloS one 20140904 9

A growing variety of "genotype-by-sequencing" (GBS) methods use restriction enzymes and high throughput DNA sequencing to generate data for a subset of genomic loci, allowing the simultaneous discovery and genotyping of thousands of polymorphisms in a set of multiplexed samples. We evaluated a "double-digest" restriction-site associated DNA sequencing (ddRAD-seq) protocol by 1) comparing results for a zebra finch (Taeniopygia guttata) sample with in silico predictions from the zebra finch refere ...[more]

PMID: 25188270

Similar Datasets

Project description:Whole-genome amplification by multiple displacement amplification (MDA) is a promising technique to enable the use of samples with only limited amount of DNA for the construction of RAD-seq libraries. Previous work has shown that, when the amount of DNA used in the MDA reaction is large, double-digest RAD-seq (ddRAD) libraries prepared with amplified genomic DNA result in data that are indistinguishable from libraries prepared directly from genomic DNA. Based on this observation, here we evaluate the quality of ddRAD libraries prepared from MDA-amplified genomic DNA when the amount of input genomic DNA and the coverage obtained for samples is variable. By simultaneously preparing libraries for five species of weevils (Coleoptera, Curculionidae), we also evaluate the likelihood that potential contaminants will be encountered in the assembled dataset. Overall, our results indicate that MDA may not be able to rescue all samples with small amounts of DNA, but it does produce ddRAD libraries adequate for studies of phylogeography and population genetics even when conditions are not optimal. We find that MDA makes it harder to predict the number of loci that will be obtained for a given sequencing effort, with some samples behaving like traditional libraries and others yielding fewer loci than expected. This seems to be caused both by stochastic and deterministic effects during amplification. Further, the reduction in loci is stronger in libraries with lower amounts of template DNA for the MDA reaction. Even though a few samples exhibit substantial levels of contamination in raw reads, the effect is very small in the final dataset, suggesting that filters imposed during dataset assembly are important in removing contamination. Importantly, samples with strong signs of contamination and biases in heterozygosity were also those with fewer loci shared in the final dataset, suggesting that stringent filtering of samples with significant amounts of missing data is important when assembling data derived from MDA-amplified genomic DNA. Overall, we find that the combination of MDA and ddRAD results in high-quality datasets for population genetics as long as the sequence data is properly filtered during assembly.

Dataset Information

Amplification biases and consistent recovery of loci in a double-digest RAD-seq protocol.

Publications

Amplification biases and consistent recovery of loci in a double-digest RAD-seq protocol.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets