Deep sequencing to reveal new variants in pooled DNA samples
Identifieur interne : 002898 ( Main/Exploration ); précédent : 002897; suivant : 002899Deep sequencing to reveal new variants in pooled DNA samples
Auteurs : Astrid A. Out [Pays-Bas] ; Ivonne J. H. M. Van Minderhout [Pays-Bas] ; Jelle J. Goeman [Pays-Bas] ; Yavuz Ariyurek [Pays-Bas] ; Stephan Ossowski [Allemagne] ; Korbinian Schneeberger [Allemagne] ; Detlef Weigel [Allemagne] ; Michiel Van Galen [Pays-Bas] ; Peter E. M. Taschner [Pays-Bas] ; Carli M. J. Tops [Pays-Bas] ; Martijn H. Breuning [Pays-Bas] ; Gert-Jan B. Van Ommen [Pays-Bas] ; Johan T. Den Dunnen [Pays-Bas] ; Peter Devilee [Pays-Bas] ; Frederik J. Hes [Pays-Bas]Source :
- Human Mutation [ 1059-7794 ] ; 2009-12.
English descriptors
- Teeft :
- Allele, Allele frequencies, Allele frequency, Allele frequency estimates, Amplification bias, Attractive method, Blank lrps, Breast cancer, Breast cancer patients, Candidate gene, Candidate genes, Chastity, Chastity threshold, Clinical genetics, Color figure, Common variants, Concentration measurement, Coverage depth, Dbsnp, Development core team, Different allele frequencies, Different error proportions, Different settings, Doubleton, Eland, Error model, Error proportion, Error proportion qnull, Error rate, False negatives, False positives, First pool, Fragment ends, Genet, Genome, Genomic, Haploid redundancy, Haplotype reconstruction, Human genome, Human mutation, Idaho technology, Illumina, Illumina data, Illumina sequencing, Illumina sequencing results, Illumina technology, Individual samples, Insertion allele, Leiden, Leiden university, Local coverage depth, Minor allele frequencies, Minor allele frequency, Mismatch, Multiple testing, Mutation, Mutyh, Mutyh gene, Nonunique regions, Nucleic acids, Null hypothesis, Online issue, Optimal fraction, Parallel sequencing, Pearson correlation coefficient, Percentile, Pool size, Qnull, Quality threshold, Rare allele, Rare variant detection, Rare variants, Reference sequence, Reliable detection, Repetitive regions, Sanger, Sanger data, Sanger sequencing, Sanger sequencing data, Second pool, Sequence coverage uniformity, Sequence depth, Sequence variant, Sequence variants, Sequencing, Sequencing errors, Shore pipeline, Single nucleotide polymorphisms, Singleton, Singleton variant, Singleton variants, Statistical power, Supp, True positives, Unique variants, Variant, Variant detection.
Abstract
We evaluated massive parallel sequencing and long‐range PCR (LRP) for rare variant detection and allele frequency estimation in pooled DNA samples. Exons 2 to 16 of the MUTYH gene were analyzed in breast cancer patients with Illumina's (Solexa) technology. From a pool of 287 genomic DNA samples we generated a single LRP product, while the same LRP was performed on 88 individual samples and the resulting products then pooled. Concentrations of constituent samples were measured with fluorimetry for genomic DNA and high‐resolution melting curve analysis (HR‐MCA) for LRP products. Illumina sequencing results were compared to Sanger sequencing data of individual samples. Correlation between allele frequencies detected by both methods was poor in the first pool, presumably because the genomic samples amplified unequally in the LRP, due to DNA quality variability. In contrast, allele frequencies correlated well in the second pool, in which all expected alleles at a frequency of 1% and higher were reliably detected, plus the majority of singletons (0.6% allele frequency). We describe custom bioinformatics and statistics to optimize detection of rare variants and to estimate required sequencing depth. Our results provide directions for designing high‐throughput analyses of candidate genes. Hum Mutat 30:1–10, 2009. © 2009 Wiley‐Liss, Inc.
Url:
DOI: 10.1002/humu.21122
Affiliations:
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Out</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Clinical Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Pays-Bas</country></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Correspondence address: Department of Clinical Genetics, Leiden University Medical Center, Post zone S6‐P, P.O. Box 9600, 2300 RC, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Van Minderhout, Ivonne J H M" sort="Van Minderhout, Ivonne J H M" uniqKey="Van Minderhout I" first="Ivonne J. H. M." last="Van Minderhout">Ivonne J. H. M. 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Goeman</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Medical Statistics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Ariyurek, Yavuz" sort="Ariyurek, Yavuz" uniqKey="Ariyurek Y" first="Yavuz" last="Ariyurek">Yavuz Ariyurek</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Human Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Leiden Genome Technology Center, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Ossowski, Stephan" sort="Ossowski, Stephan" uniqKey="Ossowski S" first="Stephan" last="Ossowski">Stephan Ossowski</name><affiliation wicri:level="3"><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen</wicri:regionArea><placeName><region type="land" nuts="1">Bade-Wurtemberg</region><region type="district" nuts="2">District de Tübingen</region><settlement type="city">Tübingen</settlement></placeName></affiliation></author><author><name sortKey="Schneeberger, Korbinian" sort="Schneeberger, Korbinian" uniqKey="Schneeberger K" first="Korbinian" last="Schneeberger">Korbinian Schneeberger</name><affiliation wicri:level="3"><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen</wicri:regionArea><placeName><region type="land" nuts="1">Bade-Wurtemberg</region><region type="district" nuts="2">District de Tübingen</region><settlement type="city">Tübingen</settlement></placeName></affiliation></author><author><name sortKey="Weigel, Detlef" sort="Weigel, Detlef" uniqKey="Weigel D" first="Detlef" last="Weigel">Detlef Weigel</name><affiliation wicri:level="3"><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen</wicri:regionArea><placeName><region type="land" nuts="1">Bade-Wurtemberg</region><region type="district" nuts="2">District de Tübingen</region><settlement type="city">Tübingen</settlement></placeName></affiliation></author><author><name sortKey="Van Galen, Michiel" sort="Van Galen, Michiel" uniqKey="Van Galen M" first="Michiel" last="Van Galen">Michiel Van Galen</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Human Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Leiden Genome Technology Center, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Taschner, Peter E M" sort="Taschner, Peter E M" uniqKey="Taschner P" first="Peter E. M." last="Taschner">Peter E. M. Taschner</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Human Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Leiden Genome Technology Center, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Tops, Carli M J" sort="Tops, Carli M J" uniqKey="Tops C" first="Carli M. J." last="Tops">Carli M. J. Tops</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Clinical Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Breuning, Martijn H" sort="Breuning, Martijn H" uniqKey="Breuning M" first="Martijn H." last="Breuning">Martijn H. Breuning</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Clinical Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Van Ommen, Gert An B" sort="Van Ommen, Gert An B" uniqKey="Van Ommen G" first="Gert-Jan B." last="Van Ommen">Gert-Jan B. Van Ommen</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Human Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Leiden Genome Technology Center, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Den Dunnen, Johan T" sort="Den Dunnen, Johan T" uniqKey="Den Dunnen J" first="Johan T." last="Den Dunnen">Johan T. Den Dunnen</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Clinical Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Human Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Leiden Genome Technology Center, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Devilee, Peter" sort="Devilee, Peter" uniqKey="Devilee P" first="Peter" last="Devilee">Peter Devilee</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Human Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pathology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Hes, Frederik J" sort="Hes, Frederik J" uniqKey="Hes F" first="Frederik J." last="Hes">Frederik J. Hes</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Clinical Genetics, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Human Mutation</title><title level="j" type="alt">HUMAN MUTATION</title><idno type="ISSN">1059-7794</idno><idno type="eISSN">1098-1004</idno><imprint><biblScope unit="vol">30</biblScope><biblScope unit="issue">12</biblScope><biblScope unit="page" from="1703">1703</biblScope><biblScope unit="page" to="1712">1712</biblScope><biblScope unit="page-count">10</biblScope><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><date type="published" when="2009-12">2009-12</date></imprint><idno type="ISSN">1059-7794</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1059-7794</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Allele</term><term>Allele frequencies</term><term>Allele frequency</term><term>Allele frequency estimates</term><term>Amplification bias</term><term>Attractive method</term><term>Blank lrps</term><term>Breast cancer</term><term>Breast cancer patients</term><term>Candidate gene</term><term>Candidate genes</term><term>Chastity</term><term>Chastity threshold</term><term>Clinical genetics</term><term>Color figure</term><term>Common variants</term><term>Concentration measurement</term><term>Coverage depth</term><term>Dbsnp</term><term>Development core team</term><term>Different allele frequencies</term><term>Different error proportions</term><term>Different settings</term><term>Doubleton</term><term>Eland</term><term>Error model</term><term>Error proportion</term><term>Error proportion qnull</term><term>Error rate</term><term>False negatives</term><term>False positives</term><term>First pool</term><term>Fragment ends</term><term>Genet</term><term>Genome</term><term>Genomic</term><term>Haploid redundancy</term><term>Haplotype reconstruction</term><term>Human genome</term><term>Human mutation</term><term>Idaho technology</term><term>Illumina</term><term>Illumina data</term><term>Illumina sequencing</term><term>Illumina sequencing results</term><term>Illumina technology</term><term>Individual samples</term><term>Insertion allele</term><term>Leiden</term><term>Leiden university</term><term>Local coverage depth</term><term>Minor allele frequencies</term><term>Minor allele frequency</term><term>Mismatch</term><term>Multiple testing</term><term>Mutation</term><term>Mutyh</term><term>Mutyh gene</term><term>Nonunique regions</term><term>Nucleic acids</term><term>Null hypothesis</term><term>Online issue</term><term>Optimal fraction</term><term>Parallel sequencing</term><term>Pearson correlation coefficient</term><term>Percentile</term><term>Pool size</term><term>Qnull</term><term>Quality threshold</term><term>Rare allele</term><term>Rare variant detection</term><term>Rare variants</term><term>Reference sequence</term><term>Reliable detection</term><term>Repetitive regions</term><term>Sanger</term><term>Sanger data</term><term>Sanger sequencing</term><term>Sanger sequencing data</term><term>Second pool</term><term>Sequence coverage uniformity</term><term>Sequence depth</term><term>Sequence variant</term><term>Sequence variants</term><term>Sequencing</term><term>Sequencing errors</term><term>Shore pipeline</term><term>Single nucleotide polymorphisms</term><term>Singleton</term><term>Singleton variant</term><term>Singleton variants</term><term>Statistical power</term><term>Supp</term><term>True positives</term><term>Unique variants</term><term>Variant</term><term>Variant detection</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">We evaluated massive parallel sequencing and long‐range PCR (LRP) for rare variant detection and allele frequency estimation in pooled DNA samples. Exons 2 to 16 of the MUTYH gene were analyzed in breast cancer patients with Illumina's (Solexa) technology. From a pool of 287 genomic DNA samples we generated a single LRP product, while the same LRP was performed on 88 individual samples and the resulting products then pooled. Concentrations of constituent samples were measured with fluorimetry for genomic DNA and high‐resolution melting curve analysis (HR‐MCA) for LRP products. Illumina sequencing results were compared to Sanger sequencing data of individual samples. Correlation between allele frequencies detected by both methods was poor in the first pool, presumably because the genomic samples amplified unequally in the LRP, due to DNA quality variability. In contrast, allele frequencies correlated well in the second pool, in which all expected alleles at a frequency of 1% and higher were reliably detected, plus the majority of singletons (0.6% allele frequency). We describe custom bioinformatics and statistics to optimize detection of rare variants and to estimate required sequencing depth. Our results provide directions for designing high‐throughput analyses of candidate genes. Hum Mutat 30:1–10, 2009. © 2009 Wiley‐Liss, Inc.</div></front></TEI><affiliations><list><country><li>Allemagne</li><li>Pays-Bas</li></country><region><li>Bade-Wurtemberg</li><li>District de Tübingen</li><li>Hollande-Méridionale</li></region><settlement><li>Leyde</li><li>Tübingen</li></settlement></list><tree><country name="Pays-Bas"><region name="Hollande-Méridionale"><name sortKey="Out, Astrid A" sort="Out, Astrid A" uniqKey="Out A" first="Astrid A." last="Out">Astrid A. Out</name></region><name sortKey="Ariyurek, Yavuz" sort="Ariyurek, Yavuz" uniqKey="Ariyurek Y" first="Yavuz" last="Ariyurek">Yavuz Ariyurek</name><name sortKey="Ariyurek, Yavuz" sort="Ariyurek, Yavuz" uniqKey="Ariyurek Y" first="Yavuz" last="Ariyurek">Yavuz Ariyurek</name><name sortKey="Breuning, Martijn H" sort="Breuning, Martijn H" uniqKey="Breuning M" first="Martijn H." last="Breuning">Martijn H. Breuning</name><name sortKey="Den Dunnen, Johan T" sort="Den Dunnen, Johan T" uniqKey="Den Dunnen J" first="Johan T." last="Den Dunnen">Johan T. Den Dunnen</name><name sortKey="Den Dunnen, Johan T" sort="Den Dunnen, Johan T" uniqKey="Den Dunnen J" first="Johan T." last="Den Dunnen">Johan T. Den Dunnen</name><name sortKey="Den Dunnen, Johan T" sort="Den Dunnen, Johan T" uniqKey="Den Dunnen J" first="Johan T." last="Den Dunnen">Johan T. Den Dunnen</name><name sortKey="Devilee, Peter" sort="Devilee, Peter" uniqKey="Devilee P" first="Peter" last="Devilee">Peter Devilee</name><name sortKey="Devilee, Peter" sort="Devilee, Peter" uniqKey="Devilee P" first="Peter" last="Devilee">Peter Devilee</name><name sortKey="Goeman, Jelle J" sort="Goeman, Jelle J" uniqKey="Goeman J" first="Jelle J." last="Goeman">Jelle J. Goeman</name><name sortKey="Hes, Frederik J" sort="Hes, Frederik J" uniqKey="Hes F" first="Frederik J." last="Hes">Frederik J. Hes</name><name sortKey="Out, Astrid A" sort="Out, Astrid A" uniqKey="Out A" first="Astrid A." last="Out">Astrid A. Out</name><name sortKey="Out, Astrid A" sort="Out, Astrid A" uniqKey="Out A" first="Astrid A." last="Out">Astrid A. Out</name><name sortKey="Taschner, Peter E M" sort="Taschner, Peter E M" uniqKey="Taschner P" first="Peter E. M." last="Taschner">Peter E. M. Taschner</name><name sortKey="Taschner, Peter E M" sort="Taschner, Peter E M" uniqKey="Taschner P" first="Peter E. M." last="Taschner">Peter E. M. Taschner</name><name sortKey="Tops, Carli M J" sort="Tops, Carli M J" uniqKey="Tops C" first="Carli M. J." last="Tops">Carli M. J. Tops</name><name sortKey="Van Galen, Michiel" sort="Van Galen, Michiel" uniqKey="Van Galen M" first="Michiel" last="Van Galen">Michiel Van Galen</name><name sortKey="Van Galen, Michiel" sort="Van Galen, Michiel" uniqKey="Van Galen M" first="Michiel" last="Van Galen">Michiel Van Galen</name><name sortKey="Van Minderhout, Ivonne J H M" sort="Van Minderhout, Ivonne J H M" uniqKey="Van Minderhout I" first="Ivonne J. H. M." last="Van Minderhout">Ivonne J. H. M. Van Minderhout</name><name sortKey="Van Ommen, Gert An B" sort="Van Ommen, Gert An B" uniqKey="Van Ommen G" first="Gert-Jan B." last="Van Ommen">Gert-Jan B. Van Ommen</name><name sortKey="Van Ommen, Gert An B" sort="Van Ommen, Gert An B" uniqKey="Van Ommen G" first="Gert-Jan B." last="Van Ommen">Gert-Jan B. Van Ommen</name></country><country name="Allemagne"><region name="Bade-Wurtemberg"><name sortKey="Ossowski, Stephan" sort="Ossowski, Stephan" uniqKey="Ossowski S" first="Stephan" last="Ossowski">Stephan Ossowski</name></region><name sortKey="Schneeberger, Korbinian" sort="Schneeberger, Korbinian" uniqKey="Schneeberger K" first="Korbinian" last="Schneeberger">Korbinian Schneeberger</name><name sortKey="Weigel, Detlef" sort="Weigel, Detlef" uniqKey="Weigel D" first="Detlef" last="Weigel">Detlef Weigel</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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