Next Generation Sequencing of Pooled Samples: Guideline for Variants’ Filtering

Anand, Santosh; Mangano, Eleonora; Barizzone, Nadia; Bordoni, Roberta; Sorosina, Melissa; Clarelli, Ferdinando; Corrado, Lucia; Boneschi, Filippo Martinelli; D’Alfonso, Sandra; Bellis, Gianluca De

doi:10.1038/srep33735

Cited by 92 publications

(91 citation statements)

References 26 publications

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“…We investigated genomewide patterns of allele frequency change using clinal variation based on a filtered set of ancestry‐informative SNPs, chosen to differ in absolute allele frequency difference (>0.2) and with opposite alleles as the most common allele in each subspecies (see Methods for further details). Pool sequencing is a cost effective method for obtaining allele frequencies across a large number of genetic markers (Anand et al., ). However, error in estimates of allele frequencies associated with each individual SNP can be high due to moderate coverage and limited number of individuals incorporated in each pool in our data set.…”

Section: Resultsmentioning

confidence: 99%

A genomic map of clinal variation across the European rabbit hybrid zone

et al. 2018

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Speciation is a process proceeding from weak to complete reproductive isolation. In this continuum, naturally hybridizing taxa provide a promising avenue for revealing the genetic changes associated with the incipient stages of speciation. To identify such changes between two subspecies of rabbits that display partial reproductive isolation, we studied patterns of allele frequency change across their hybrid zone using whole-genome sequencing. To connect levels and patterns of genetic differentiation with phenotypic manifestations of subfertility in hybrid rabbits, we further investigated patterns of gene expression in testis. Geographic cline analysis revealed 253 regions characterized by steep changes in allele frequency across their natural region of contact. This catalog of regions is likely to be enriched for loci implicated in reproductive barriers and yielded several insights into the evolution of hybrid dysfunction in rabbits: (i) incomplete reproductive isolation is likely governed by the effects of many loci, (ii) protein-protein interaction analysis suggest that genes within these loci interact more than expected by chance, (iii) regulatory variation is likely the primary driver of incompatibilities, and (iv) large chromosomal rearrangements appear not to be a major mechanism underlying incompatibilities or promoting isolation in the face of gene flow. We detected extensive misregulation of gene expression in testis of hybrid males, but not a statistical overrepresentation of differentially expressed genes in candidate regions. Our results also did not support an X chromosome-wide disruption of expression as observed in mice and cats, suggesting variation in the mechanistic basis of hybrid male reduced fertility among mammals.

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Section: Resultsmentioning

confidence: 99%

A genomic map of clinal variation across the European rabbit hybrid zone

et al. 2018

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“…One of the main benefits of Pool‐seq is the generation of vast amounts of SNPs per genome for entire populations, with the prospect of improving power of population detection and delineation (Anand et al, ). However, average differentiation based on the previously identified SNPs was inflated compared to average estimates based on all Pool‐seq SNPs in the introduced population pair, but not in the natural population pair (Table ).…”

Section: Discussionmentioning

confidence: 99%

Exploring a Pool‐seq‐only approach for gaining population genomic insights in nonmodel species

Kurland

Wheat

Celorio‐Mancera

et al. 2019

Ecology and Evolution

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Developing genomic insights is challenging in nonmodel species for which resources are often scarce and prohibitively costly. Here, we explore the potential of a recently established approach using Pool‐seq data to generate a de novo genome assembly for mining exons, upon which Pool‐seq data are used to estimate population divergence and diversity. We do this for two pairs of sympatric populations of brown trout (Salmo trutta): one naturally sympatric set of populations and another pair of populations introduced to a common environment. We validate our approach by comparing the results to those from markers previously used to describe the populations (allozymes and individual‐based single nucleotide polymorphisms [SNPs]) and from mapping the Pool‐seq data to a reference genome of the closely related Atlantic salmon (Salmo salar). We find that genomic differentiation (FST) between the two introduced populations exceeds that of the naturally sympatric populations (FST = 0.13 and 0.03 between the introduced and the naturally sympatric populations, respectively), in concordance with estimates from the previously used SNPs. The same level of population divergence is found for the two genome assemblies, but estimates of average nucleotide diversity differ (trueπ¯ ≈ 0.002 and trueπ¯ ≈ 0.001 when mapping to S. trutta and S. salar, respectively), although the relationships between population values are largely consistent. This discrepancy might be attributed to biases when mapping to a haploid condensed assembly made of highly fragmented read data compared to using a high‐quality reference assembly from a divergent species. We conclude that the Pool‐seq‐only approach can be suitable for detecting and quantifying genome‐wide population differentiation, and for comparing genomic diversity in populations of nonmodel species where reference genomes are lacking.

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“…Pooling samples before library preparations, also called "poolseq", can be used for projects with hundreds of samples and if tracing back individual samples is not relevant for the research question at hand (Himmelbach et al, 2014;Anand et al, 2016). This strategy is useful for the identification of variable regions between populations, especially when population sampling must be higher than what the budget allows for sequencing as individual libraries (Neethiraj et al, 2017).…”

Section: Amplificationmentioning

confidence: 99%

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project

et al. 2020

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High-throughput DNA sequencing techniques enable time-and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing effort on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing coverage. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth. Moreover, it has proven to produce powerful, large multi-locus DNA sequence datasets suitable for phylogenetic analyses. However, target capture requires careful considerations, which may greatly affect the success of experiments. Here we provide a simple flowchart for designing phylogenomic target capture experiments. We discuss necessary decisions from the identification of target loci to the final bioinformatic processing of sequence data. We outline challenges and solutions related to the taxonomic scope, sample quality, and available genomic resources of target capture projects. We hope this review will serve as a useful roadmap for designing and carrying out successful phylogenetic target capture studies.

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Next Generation Sequencing of Pooled Samples: Guideline for Variants’ Filtering

Cited by 92 publications

References 26 publications

A genomic map of clinal variation across the European rabbit hybrid zone

A genomic map of clinal variation across the European rabbit hybrid zone

Exploring a Pool‐seq‐only approach for gaining population genomic insights in nonmodel species

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project

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