Finding the right coverage: the impact of coverage and sequence quality on single nucleotide polymorphism genotyping error rates

Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction‐enzyme‐based methods remain largely unknown....

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Vydáno v:	Molecular ecology resources Ročník 16; číslo 4; s. 966 - 978
Hlavní autoři:	Fountain, Emily D., Pauli, Jonathan N., Reid, Brendan N., Palsbøll, Per J., Peery, M. Zachariah
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	England Blackwell Publishing Ltd 01.07.2016 Wiley Subscription Services, Inc
Témata:	Animals Choloepus Choloepus hoffmanni data collection Datasets ddRAD Diagnostic Errors DNA libraries DNA Restriction Enzymes - metabolism Economic models Enzymes Genetic markers genotyping error genotyping errors Genotyping Techniques - methods High-Throughput Nucleotide Sequencing loci Mendelian incompatibility next-generation sequencing Offspring parentage Polymorphism, Single Nucleotide Quality Sequence Analysis, DNA single nucleotide polymorphism Xenarthra - classification Xenarthra - genetics ddRAD genotyping error next-generation sequencing single nucleotide polymorphism Mendelian incompatibility
ISSN:	1755-098X, 1755-0998
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Abstract	Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction‐enzyme‐based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother–offspring dyads of Hoffman's two‐toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference‐aligned and de novo‐assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo‐assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference‐aligned and de novo‐assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference‐aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo‐assembled data sets. We observed approximately 10‐ and 13‐fold declines in the number of loci sampled in the reference‐aligned and de novo‐assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade‐off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction‐enzyme‐based SNP studies.
AbstractList	Restriction-enzyme-based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction-enzyme-based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother-offspring dyads of Hoffman's two-toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference-aligned and de novo-assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo-assembled data sets. For example, coverage greater than or equal to 5 yielded median genotyping error rates of greater than or equal to 0.03 and greater than or equal to 0.11 in reference-aligned and de novo-assembled data sets, respectively. Genotyping error rates declined to less than or equal to 0.01 in reference-aligned data sets with a coverage greater than or equal to 30, but remained greater than or equal to 0.04 in the de novo-assembled data sets. We observed approximately 10- and 13-fold declines in the number of loci sampled in the reference-aligned and de novo-assembled data sets when coverage was increased from greater than or equal to 5 to greater than or equal to 30 at quality score greater than or equal to 30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade-off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction-enzyme-based SNP studies. Restriction-enzyme-based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction-enzyme-based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother-offspring dyads of Hoffman's two-toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference-aligned and de novo-assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo-assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference-aligned and de novo-assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference-aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo-assembled data sets. We observed approximately 10- and 13-fold declines in the number of loci sampled in the reference-aligned and de novo-assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade-off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction-enzyme-based SNP studies. Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism ( SNP ) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNP s derived from restriction‐enzyme‐based methods remain largely unknown. Here, we estimated genotyping error rates in SNP s genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother–offspring dyads of Hoffman's two‐toed sloth ( Choloepus hoffmanni ) across a range of coverage and sequence quality criteria, for both reference‐aligned and de novo ‐assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo ‐assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference‐aligned and de novo ‐assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference‐aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo‐ assembled data sets. We observed approximately 10‐ and 13‐fold declines in the number of loci sampled in the reference‐aligned and de novo ‐assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade‐off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction‐enzyme‐based SNP studies.
Author	Pauli, Jonathan N. Palsbøll, Per J. Peery, M. Zachariah Reid, Brendan N. Fountain, Emily D.
Author_xml	– sequence: 1 givenname: Emily D. surname: Fountain fullname: Fountain, Emily D. email: efountain@wisc.edu organization: Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, WI, 53706, Madison, USA – sequence: 2 givenname: Jonathan N. surname: Pauli fullname: Pauli, Jonathan N. organization: Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, WI, 53706, Madison, USA – sequence: 3 givenname: Brendan N. surname: Reid fullname: Reid, Brendan N. organization: Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, WI, 53706, Madison, USA – sequence: 4 givenname: Per J. surname: Palsbøll fullname: Palsbøll, Per J. organization: Marine Evolution and Conservation, Groningen Institute of Evolutionary Life Sciences, University of Groningen, AG, 9747, Groningen, The Netherlands – sequence: 5 givenname: M. Zachariah surname: Peery fullname: Peery, M. Zachariah organization: Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, WI, 53706, Madison, USA
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Keywords	ddRAD genotyping error next-generation sequencing single nucleotide polymorphism Mendelian incompatibility
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Notes	ArticleID:MEN12519 NSF - No. #1257535 ark:/67375/WNG-SZ16TVSL-Q istex:17B5285FEB29D7DA6230452FBB4162104BD6F60D Table S1 Total number of raw and retained reads by individuals, sample quality and the sequencing library for quality score ≥10. Table S2 Total number of raw and retained reads by individuals, sample quality and the sequencing library for quality score ≥20. Table S3 Total number of raw and retained reads by individuals, sample quality and the sequencing library for quality score ≥30. Fig. S1 Figure of median genotyping error for reference-aligned full reads using all 16 dyads vs. dataset with the three low-quality dyads removed. Fig. S2 Reference-aligned full dataset changes relative to that observed at minimum coverage (≥5) and quality score (≥10). Fig. S3de novo-assembled dataset changes relative to that observed at minimum coverage (≥5) and quality score (≥10). Fig. S4 Figure of median genotyping error for de novo-assembled dataset using all 16 dyads vs. dataset with the three low-quality dyads removed. Fig. S5 rxstacks corrected dataset changes relative to that observed at minimum coverage (≥5) and quality score (≥10) for reference-aligned full and de novo-assembled datasets.Appendix S1 Detailed methods for laboratory protocols, de novo assembly parameter tests and parentage assignments. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
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Snippet	Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in... Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism ( SNP ) loci in nonmodel organisms. However,... Restriction-enzyme-based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in...
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SubjectTerms	Animals Choloepus Choloepus hoffmanni data collection Datasets ddRAD Diagnostic Errors DNA libraries DNA Restriction Enzymes - metabolism Economic models Enzymes Genetic markers genotyping error genotyping errors Genotyping Techniques - methods High-Throughput Nucleotide Sequencing loci Mendelian incompatibility next-generation sequencing Offspring parentage Polymorphism, Single Nucleotide Quality Sequence Analysis, DNA single nucleotide polymorphism Xenarthra - classification Xenarthra - genetics
Title	Finding the right coverage: the impact of coverage and sequence quality on single nucleotide polymorphism genotyping error rates
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