The Next Generation of Molecular Markers From Massively Parallel Sequencing of Pooled DNA Samples

Abstract

Next generation sequencing (NGS) is about to revolutionize genetic analysis. Currently NGS techniques are mainly used to sequence individual genomes. Due to the high sequence coverage required, the costs for population-scale analyses are still too high to allow an extension to nonmodel organisms. Here, we show that NGS of pools of individuals is often more effective in SNP discovery and provides more accurate allele frequency estimates, even when taking sequencing errors into account. We modify the population genetic estimators Tajima's p and Watterson's u to obtain unbiased estimates from NGS pooling data. Given the same sequencing effort, the resulting estimators often show a better performance than those obtained from individual sequencing. Although our analysis also shows that NGS of pools of individuals will not be preferable under all circumstances, it provides a cost-effective approach to estimate allele frequencies on a genome-wide scale. N EXT generation sequencing (NGS) is about to revolutionize biology. Through a massive parallelization, NGS provides an enormous number of reads, which permits sequencing of entire genomes at a fraction of the costs for Sanger sequencing. Hence, for the first time it has become feasible to obtain the complete genomic sequence for a large number of individuals. For several organisms, including humans, Drosophila melanogaster, and Arabidopsis thaliana, large resequencing projects are well on their way. Nevertheless, despite the enormous cost reduction, genome sequencing on a population scale is still out of reach for the budget of most laboratories. The extraction of as much statistical information as possible at cost as low as possible has therefore already attracted considerable interest. See, for instance, Jiang et al. Current genome-wide resequencing projects collect the sequences individual by individual. To obtain full coverage of the entire genome and to have high confidence that all heterozygous sites were discovered, it is required that genomes are sequenced at a sufficiently high coverage. As many of the reads provide only redundant information, cost could be reduced by a more effective sampling strategy.In this report, we explore the potential of DNA pooling to provide a more cost-effective approach for SNP discovery and genome-wide population genetics. Sequencing a large pool of individuals simultaneously keeps the number of redundant DNA reads low and provides thus an economic alternative to the sequencing of individual genomes. On the other hand, more care has to be taken to establish an appropriate control of sequencing errors. Obviously haplotype information is not available from pooling experiments, but this will often be outweighed by the increased accuracy in population genetic inference.Focusing on biallelic loci, our analysis shows that with sufficiently large pool sizes, pooling usually outperforms the separate sequencing of individuals, both for estimating allele frequencies and for inference of population genetic parameters. When sequencing...

Multi-locus analysis of genomic time series data from experimental evolution

“…(Indeed, there is a discussion on the utility and power of pooled sequencing [37,[43][44][45].) The same multi-locus model underlying our approach can be applied to develop a method for analyzing haplotypic time series data, and we will explore incorporating such an extension into our method.…”

“…A sexually reproducing population of N diploid individuals is evolved in discrete, non-overlapping generations. Pooled DNA sequencing [37,38] is performed T times at generations t 1 < t 2 < Á Á Á < t T . At each segregating site in the resulting data set, we assume that there are two alleles, denoted A 0 and A 1 .…”

Applications of next generation sequencing in molecular ecology of non-model organisms

“…SNPs (located in the coding region or in the UTRs) can also be discovered using many different approaches (for an updated list of specific software available for various applications see: http://seqanswers.com/wiki/ Special:BrowseData/Bioinformatics_application). Both data from individually sequenced samples or samples of pooled individuals are useful to this end (Futschik and Schlö tterer, 2010).…”

“…The trade off between sequence depth of individual samples and number of samples sequenced also needs to be considered, as the calculations of allele frequencies and detection of low-frequency SNPs will be severely hampered if there is insufficient coverage of individual SNPs. It should also be noted that for SNP detection and estimation of population biology parameters, sequencing pools of individuals may be more effective than individual tagging of sequences (Futschik and Schlö tterer, 2010).…”

Understanding and monitoring the consequences of human impacts on intraspecific variation

“…These approaches do not require a pre‐existing reference genome, although availability of such a genome allows for more accurate calls for SNPs and a better ability to infer selection (discussed below). Pool‐seq (Futschik & Schlotterer, 2010) combines DNA from multiple individuals into a single sequencing run, which greatly reduces the cost of obtaining allele frequency data but sacrifices information about the linkage among genetic polymorphisms. Finally, RNA‐seq can be used to measure expression differences across thousands of genes without relying on a priori assumptions about which genes are important.…”