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Title: Accurate Allele Frequencies from Ultra-low Coverage Pool-Seq Samples in Evolve-and-Resequence Experiments

Abstract

Evolve-and-resequence (E+R) experiments leverage next-generation sequencing technology to track the allele frequency dynamics of populations as they evolve. While previous work has shown that adaptive alleles can be detected by comparing frequency trajectories from many replicate populations, this power comes at the expense of high-coverage (>100x) sequencing of many pooled samples, which can be cost-prohibitive. Here, we show that accurate estimates of allele frequencies can be achieved with very shallow sequencing depths (<5x) via inference of known founder haplotypes in small genomic windows. This technique can be used to efficiently estimate frequencies for any number of bi-allelic SNPs in populations of any model organism founded with sequenced homozygous strains. Using both experimentally-pooled and simulated samples of Drosophila melanogaster, we show that haplotype inference can improve allele frequency accuracy by orders of magnitude for up to 50 generations of recombination, and is robust to moderate levels of missing data, as well as different selection regimes. Finally, we show that a simple linear model generated from these simulations can predict the accuracy of haplotype-derived allele frequencies in other model organisms and experimental designs. To make these results broadly accessible for use in E+R experiments, we introduce HAF-pipe, an open-source software tool formore » calculating haplotype-derived allele frequencies from raw sequencing data. Ultimately, by reducing sequencing costs without sacrificing accuracy, our method facilitates E+R designs with higher replication and resolution, and thereby, increased power to detect adaptive alleles.« less

Authors:
ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [1]; ORCiD logo [3]; ORCiD logo [1]; ORCiD logo [4]
  1. Stanford Univ., CA (United States)
  2. Stanford Univ., CA (United States); Univ. of Virginia, Charlottesville, VA (United States)
  3. Univ. of Pennsylvania, Philadelphia, PA (United States)
  4. Stanford Univ., CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1591845
Grant/Contract Number:  
[AC02-05CH11231]
Resource Type:
Accepted Manuscript
Journal Name:
G3
Additional Journal Information:
[ Journal Volume: 9; Journal Issue: 12]; Journal ID: ISSN 2160-1836
Publisher:
Genetics Society of America
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; evolve-and-resequence; allele; frequencies; coverage; adaptation; haplotypes

Citation Formats

Tilk, Susanne, Bergland, Alan, Goodman, Aaron, Schmidt, Paul, Petrov, Dmitri, and Greenblum, Sharon. Accurate Allele Frequencies from Ultra-low Coverage Pool-Seq Samples in Evolve-and-Resequence Experiments. United States: N. p., 2019. Web. doi:10.1534/g3.119.400755.
Tilk, Susanne, Bergland, Alan, Goodman, Aaron, Schmidt, Paul, Petrov, Dmitri, & Greenblum, Sharon. Accurate Allele Frequencies from Ultra-low Coverage Pool-Seq Samples in Evolve-and-Resequence Experiments. United States. doi:10.1534/g3.119.400755.
Tilk, Susanne, Bergland, Alan, Goodman, Aaron, Schmidt, Paul, Petrov, Dmitri, and Greenblum, Sharon. Tue . "Accurate Allele Frequencies from Ultra-low Coverage Pool-Seq Samples in Evolve-and-Resequence Experiments". United States. doi:10.1534/g3.119.400755. https://www.osti.gov/servlets/purl/1591845.
@article{osti_1591845,
title = {Accurate Allele Frequencies from Ultra-low Coverage Pool-Seq Samples in Evolve-and-Resequence Experiments},
author = {Tilk, Susanne and Bergland, Alan and Goodman, Aaron and Schmidt, Paul and Petrov, Dmitri and Greenblum, Sharon},
abstractNote = {Evolve-and-resequence (E+R) experiments leverage next-generation sequencing technology to track the allele frequency dynamics of populations as they evolve. While previous work has shown that adaptive alleles can be detected by comparing frequency trajectories from many replicate populations, this power comes at the expense of high-coverage (>100x) sequencing of many pooled samples, which can be cost-prohibitive. Here, we show that accurate estimates of allele frequencies can be achieved with very shallow sequencing depths (<5x) via inference of known founder haplotypes in small genomic windows. This technique can be used to efficiently estimate frequencies for any number of bi-allelic SNPs in populations of any model organism founded with sequenced homozygous strains. Using both experimentally-pooled and simulated samples of Drosophila melanogaster, we show that haplotype inference can improve allele frequency accuracy by orders of magnitude for up to 50 generations of recombination, and is robust to moderate levels of missing data, as well as different selection regimes. Finally, we show that a simple linear model generated from these simulations can predict the accuracy of haplotype-derived allele frequencies in other model organisms and experimental designs. To make these results broadly accessible for use in E+R experiments, we introduce HAF-pipe, an open-source software tool for calculating haplotype-derived allele frequencies from raw sequencing data. Ultimately, by reducing sequencing costs without sacrificing accuracy, our method facilitates E+R designs with higher replication and resolution, and thereby, increased power to detect adaptive alleles.},
doi = {10.1534/g3.119.400755},
journal = {G3},
number = [12],
volume = [9],
place = {United States},
year = {2019},
month = {12}
}

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Works referenced in this record:

A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus
journal, September 2016

  • Lack, Justin B.; Lange, Jeremy D.; Tang, Alison D.
  • Molecular Biology and Evolution, Vol. 33, Issue 12
  • DOI: 10.1093/molbev/msw195

Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data
journal, March 2016


Different Trajectories of Parallel Evolution During Viral Adaptation
journal, July 1999


Investigating Natural Variation in Drosophila Courtship Song by the Evolve and Resequence Approach
journal, March 2012


CeNDR, the Caenorhabditis elegans natural diversity resource
journal, October 2016

  • Cook, Daniel E.; Zdraljevic, Stefan; Roberts, Joshua P.
  • Nucleic Acids Research, Vol. 45, Issue D1
  • DOI: 10.1093/nar/gkw893

Ancestral population reconstitution from isofemale lines as a tool for experimental evolution
journal, August 2016

  • Nouhaud, Pierre; Tobler, Ray; Nolte, Viola
  • Ecology and Evolution, Vol. 6, Issue 20
  • DOI: 10.1002/ece3.2402

Rapid seasonal evolution in innate immunity of wild Drosophila melanogaster
journal, January 2018

  • Behrman, Emily L.; Howick, Virginia M.; Kapun, Martin
  • Proceedings of the Royal Society B: Biological Sciences, Vol. 285, Issue 1870
  • DOI: 10.1098/rspb.2017.2599

Population-Genetic Inference from Pooled-Sequencing Data
journal, April 2014

  • Lynch, Michael; Bost, Darius; Wilson, Sade
  • Genome Biology and Evolution, Vol. 6, Issue 5
  • DOI: 10.1093/gbe/evu085

LDx: Estimation of Linkage Disequilibrium from High-Throughput Pooled Resequencing Data
journal, November 2012


The Many Landscapes of Recombination in Drosophila melanogaster
journal, October 2012


Quantifying Selection Acting on a Complex Trait Using Allele Frequency Time Series Data
journal, November 2011

  • Illingworth, C. J. R.; Parts, L.; Schiffels, S.
  • Molecular Biology and Evolution, Vol. 29, Issue 4
  • DOI: 10.1093/molbev/msr289

Genome-wide analysis of a long-term evolution experiment with Drosophila
journal, September 2010

  • Burke, Molly K.; Dunham, Joseph P.; Shahrestani, Parvin
  • Nature, Vol. 467, Issue 7315
  • DOI: 10.1038/nature09352

Experimental evolution reveals natural selection on standing genetic variation
journal, January 2009

  • Teotónio, Henrique; Chelo, Ivo M.; Bradić, Martina
  • Nature Genetics, Vol. 41, Issue 2
  • DOI: 10.1038/ng.289

Genome evolution and adaptation in a long-term experiment with Escherichia coli
journal, October 2009

  • Barrick, Jeffrey E.; Yu, Dong Su; Yoon, Sung Ho
  • Nature, Vol. 461, Issue 7268
  • DOI: 10.1038/nature08480

PoolHap: Inferring Haplotype Frequencies from Pooled Samples by Next Generation Sequencing
journal, January 2011


Drosophila simulans : A Species with Improved Resolution in Evolve and Resequence Studies
journal, May 2017

  • Barghi, Neda; Tobler, Raymond; Nolte, Viola
  • G3&#58; Genes|Genomes|Genetics, Vol. 7, Issue 7
  • DOI: 10.1534/g3.117.043349

Standing Genetic Variation Drives Repeatable Experimental Evolution in Outcrossing Populations of Saccharomyces cerevisiae
journal, August 2014

  • Burke, Molly K.; Liti, Gianni; Long, Anthony D.
  • Molecular Biology and Evolution, Vol. 31, Issue 12
  • DOI: 10.1093/molbev/msu256

A Guide for the Design of Evolve and Resequencing Studies
journal, November 2013

  • Kofler, Robert; Schlötterer, Christian
  • Molecular Biology and Evolution, Vol. 31, Issue 2
  • DOI: 10.1093/molbev/mst221

Reconstruction of Haplotype-Blocks Selected during Experimental Evolution
journal, October 2016

  • Franssen, Susanne U.; Barton, Nicholas H.; Schlötterer, Christian
  • Molecular Biology and Evolution, Vol. 34, Issue 1
  • DOI: 10.1093/molbev/msw210

Empirical Validation of Pooled Whole Genome Population Re-Sequencing in Drosophila melanogaster
journal, July 2012


Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes
journal, May 2015


forqs: forward-in-time simulation of recombination, quantitative traits and selection
journal, December 2013


Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
journal, October 2015


Elucidating the molecular architecture of adaptation via evolve and resequence experiments
journal, September 2015

  • Long, Anthony; Liti, Gianni; Luptak, Andrej
  • Nature Reviews Genetics, Vol. 16, Issue 10
  • DOI: 10.1038/nrg3937

Evidence of widespread selection on standing variation in Europe at height-associated SNPs
journal, August 2012

  • Turchin, Michael C.; Chiang, Charleston WK; Palmer, Cameron D.
  • Nature Genetics, Vol. 44, Issue 9
  • DOI: 10.1038/ng.2368

Maximum-parsimony haplotype frequencies inference based on a joint constrained sparse representation of pooled DNA
journal, January 2013

  • Jajamovich, Guido H.; Iliadis, Alexandros; Anastassiou, Dimitris
  • BMC Bioinformatics, Vol. 14, Issue 1
  • DOI: 10.1186/1471-2105-14-270

How does adaptation sweep through the genome? Insights from long-term selection experiments
journal, October 2012

  • Burke, Molly K.
  • Proceedings of the Royal Society B: Biological Sciences, Vol. 279, Issue 1749
  • DOI: 10.1098/rspb.2012.0799

Maximum Likelihood Estimation of Frequencies of Known Haplotypes from Pooled Sequence Data
journal, January 2013

  • Kessner, D.; Turner, T. L.; Novembre, J.
  • Molecular Biology and Evolution, Vol. 30, Issue 5
  • DOI: 10.1093/molbev/mst016

Rapid Construction of Empirical RNA Fitness Landscapes
journal, October 2010


Genomic Differentiation Between Temperate and Tropical Australian Populations of Drosophila melanogaster
journal, November 2010


Whole-Genome Resequencing of Experimental Populations Reveals Polygenic Basis of Egg-Size Variation in Drosophila melanogaster
journal, June 2015

  • Jha, Aashish R.; Miles, Cecelia M.; Lippert, Nodia R.
  • Molecular Biology and Evolution, Vol. 32, Issue 10
  • DOI: 10.1093/molbev/msv136

The Power to Detect Quantitative Trait Loci Using Resequenced, Experimentally Evolved Populations of Diploid, Sexual Organisms
journal, January 2014

  • Baldwin-Brown, James G.; Long, Anthony D.; Thornton, Kevin R.
  • Molecular Biology and Evolution, Vol. 31, Issue 4
  • DOI: 10.1093/molbev/msu048

Standing genetic variation as a major contributor to adaptation in the Virginia chicken lines selection experiment
journal, October 2015


Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines
journal, April 2014


    Works referencing / citing this record:

    Ancestral population reconstitution from isofemale lines as a tool for experimental evolution
    journal, August 2016

    • Nouhaud, Pierre; Tobler, Ray; Nolte, Viola
    • Ecology and Evolution, Vol. 6, Issue 20
    • DOI: 10.1002/ece3.2402

    Genome evolution and adaptation in a long-term experiment with Escherichia coli
    journal, October 2009

    • Barrick, Jeffrey E.; Yu, Dong Su; Yoon, Sung Ho
    • Nature, Vol. 461, Issue 7268
    • DOI: 10.1038/nature08480

    Genome-wide analysis of a long-term evolution experiment with Drosophila
    journal, September 2010

    • Burke, Molly K.; Dunham, Joseph P.; Shahrestani, Parvin
    • Nature, Vol. 467, Issue 7315
    • DOI: 10.1038/nature09352

    Evidence of widespread selection on standing variation in Europe at height-associated SNPs
    journal, August 2012

    • Turchin, Michael C.; Chiang, Charleston WK; Palmer, Cameron D.
    • Nature Genetics, Vol. 44, Issue 9
    • DOI: 10.1038/ng.2368

    Experimental evolution reveals natural selection on standing genetic variation
    journal, January 2009

    • Teotónio, Henrique; Chelo, Ivo M.; Bradić, Martina
    • Nature Genetics, Vol. 41, Issue 2
    • DOI: 10.1038/ng.289

    Elucidating the molecular architecture of adaptation via evolve and resequence experiments
    journal, September 2015

    • Long, Anthony; Liti, Gianni; Luptak, Andrej
    • Nature Reviews Genetics, Vol. 16, Issue 10
    • DOI: 10.1038/nrg3937

    forqs: forward-in-time simulation of recombination, quantitative traits and selection
    journal, December 2013


    Population-Genetic Inference from Pooled-Sequencing Data
    journal, April 2014

    • Lynch, Michael; Bost, Darius; Wilson, Sade
    • Genome Biology and Evolution, Vol. 6, Issue 5
    • DOI: 10.1093/gbe/evu085

    Quantifying Selection Acting on a Complex Trait Using Allele Frequency Time Series Data
    journal, November 2011

    • Illingworth, C. J. R.; Parts, L.; Schiffels, S.
    • Molecular Biology and Evolution, Vol. 29, Issue 4
    • DOI: 10.1093/molbev/msr289

    Maximum Likelihood Estimation of Frequencies of Known Haplotypes from Pooled Sequence Data
    journal, January 2013

    • Kessner, D.; Turner, T. L.; Novembre, J.
    • Molecular Biology and Evolution, Vol. 30, Issue 5
    • DOI: 10.1093/molbev/mst016

    A Guide for the Design of Evolve and Resequencing Studies
    journal, November 2013

    • Kofler, Robert; Schlötterer, Christian
    • Molecular Biology and Evolution, Vol. 31, Issue 2
    • DOI: 10.1093/molbev/mst221

    The Power to Detect Quantitative Trait Loci Using Resequenced, Experimentally Evolved Populations of Diploid, Sexual Organisms
    journal, January 2014

    • Baldwin-Brown, James G.; Long, Anthony D.; Thornton, Kevin R.
    • Molecular Biology and Evolution, Vol. 31, Issue 4
    • DOI: 10.1093/molbev/msu048

    Standing Genetic Variation Drives Repeatable Experimental Evolution in Outcrossing Populations of Saccharomyces cerevisiae
    journal, August 2014

    • Burke, Molly K.; Liti, Gianni; Long, Anthony D.
    • Molecular Biology and Evolution, Vol. 31, Issue 12
    • DOI: 10.1093/molbev/msu256

    Whole-Genome Resequencing of Experimental Populations Reveals Polygenic Basis of Egg-Size Variation in Drosophila melanogaster
    journal, June 2015

    • Jha, Aashish R.; Miles, Cecelia M.; Lippert, Nodia R.
    • Molecular Biology and Evolution, Vol. 32, Issue 10
    • DOI: 10.1093/molbev/msv136

    A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus
    journal, September 2016

    • Lack, Justin B.; Lange, Jeremy D.; Tang, Alison D.
    • Molecular Biology and Evolution, Vol. 33, Issue 12
    • DOI: 10.1093/molbev/msw195

    Reconstruction of Haplotype-Blocks Selected during Experimental Evolution
    journal, October 2016

    • Franssen, Susanne U.; Barton, Nicholas H.; Schlötterer, Christian
    • Molecular Biology and Evolution, Vol. 34, Issue 1
    • DOI: 10.1093/molbev/msw210

    CeNDR, the Caenorhabditis elegans natural diversity resource
    journal, October 2016

    • Cook, Daniel E.; Zdraljevic, Stefan; Roberts, Joshua P.
    • Nucleic Acids Research, Vol. 45, Issue D1
    • DOI: 10.1093/nar/gkw893

    How does adaptation sweep through the genome? Insights from long-term selection experiments
    journal, October 2012

    • Burke, Molly K.
    • Proceedings of the Royal Society B: Biological Sciences, Vol. 279, Issue 1749
    • DOI: 10.1098/rspb.2012.0799

    Rapid seasonal evolution in innate immunity of wild Drosophila melanogaster
    journal, January 2018

    • Behrman, Emily L.; Howick, Virginia M.; Kapun, Martin
    • Proceedings of the Royal Society B: Biological Sciences, Vol. 285, Issue 1870
    • DOI: 10.1098/rspb.2017.2599

    Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines
    journal, April 2014


    Rapid Construction of Empirical RNA Fitness Landscapes
    journal, October 2010


    Different Trajectories of Parallel Evolution During Viral Adaptation
    journal, July 1999


    Maximum-parsimony haplotype frequencies inference based on a joint constrained sparse representation of pooled DNA
    journal, January 2013

    • Jajamovich, Guido H.; Iliadis, Alexandros; Anastassiou, Dimitris
    • BMC Bioinformatics, Vol. 14, Issue 1
    • DOI: 10.1186/1471-2105-14-270

    Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data
    journal, March 2016


    Standing genetic variation as a major contributor to adaptation in the Virginia chicken lines selection experiment
    journal, October 2015


    The Many Landscapes of Recombination in Drosophila melanogaster
    journal, October 2012


    PoolHap: Inferring Haplotype Frequencies from Pooled Samples by Next Generation Sequencing
    journal, January 2011


    Empirical Validation of Pooled Whole Genome Population Re-Sequencing in Drosophila melanogaster
    journal, July 2012


    LDx: Estimation of Linkage Disequilibrium from High-Throughput Pooled Resequencing Data
    journal, November 2012


    Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes
    journal, May 2015


    Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
    journal, October 2015


    Drosophila simulans : A Species with Improved Resolution in Evolve and Resequence Studies
    journal, May 2017

    • Barghi, Neda; Tobler, Raymond; Nolte, Viola
    • G3&#58; Genes|Genomes|Genetics, Vol. 7, Issue 7
    • DOI: 10.1534/g3.117.043349

    Genomic Differentiation Between Temperate and Tropical Australian Populations of Drosophila melanogaster
    journal, November 2010


    Investigating Natural Variation in Drosophila Courtship Song by the Evolve and Resequence Approach
    journal, March 2012