A Framework for Variation Discovery and Genotyping Using Next-generation DNA Sequencing Data

Overview

Journal Nat Genet

Specialty Genetics

Date 2011 Apr 12

PMID 21478889

Citations 6255

Authors

Mark A DePristo

Eric Banks

Ryan Poplin

Kiran V Garimella

Jared R Maguire

Christopher Hartl

Anthony A Philippakis

Guillermo Del Angel

Manuel A Rivas

Matt Hanna

Aaron McKenna

Tim J Fennell

Andrew M Kernytsky

Andrey Y Sivachenko

Kristian Cibulskis

Stacey B Gabriel

David Altshuler

Mark J Daly

Affiliations

Soon will be listed here.

Abstract

Recent advances in sequencing technology make it possible to comprehensively catalog genetic variation in population samples, creating a foundation for understanding human disease, ancestry and evolution. The amounts of raw data produced are prodigious, and many computational steps are required to translate this output into high-quality variant calls. We present a unified analytic framework to discover and genotype variation among multiple samples simultaneously that achieves sensitive and specific results across five sequencing technologies and three distinct, canonical experimental designs. Our process includes (i) initial read mapping; (ii) local realignment around indels; (iii) base quality score recalibration; (iv) SNP discovery and genotyping to find all potential variants; and (v) machine learning to separate true segregating variation from machine artifacts common to next-generation sequencing technologies. We here discuss the application of these tools, instantiated in the Genome Analysis Toolkit, to deep whole-genome, whole-exome capture and multi-sample low-pass (∼4×) 1000 Genomes Project datasets.

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References

Ning Z, Cox A, Mullikin J . SSAHA: a fast search method for large DNA databases. Genome Res. 2001; 11(10):1725-9. PMC: 311141. DOI: 10.1101/gr.194201. View

Freudenberg-Hua Y, Freudenberg J, Kluck N, Cichon S, Propping P, Nothen M . Single nucleotide variation analysis in 65 candidate genes for CNS disorders in a representative sample of the European population. Genome Res. 2003; 13(10):2271-6. PMC: 403700. DOI: 10.1101/gr.1299703. View

Dohm J, Lottaz C, Borodina T, Himmelbauer H . Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res. 2008; 36(16):e105. PMC: 2532726. DOI: 10.1093/nar/gkn425. View

Li R, Yu C, Li Y, Lam T, Yiu S, Kristiansen K . SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009; 25(15):1966-7. DOI: 10.1093/bioinformatics/btp336. View

Hoberman R, Dias J, Ge B, Harmsen E, Mayhew M, Verlaan D . A probabilistic approach for SNP discovery in high-throughput human resequencing data. Genome Res. 2009; 19(9):1542-52. PMC: 2752119. DOI: 10.1101/gr.092072.109. View

Ewing B, Green P . Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998; 8(3):186-94. View

Mokry M, Feitsma H, Nijman I, de Bruijn E, van der Zaag P, Guryev V . Accurate SNP and mutation detection by targeted custom microarray-based genomic enrichment of short-fragment sequencing libraries. Nucleic Acids Res. 2010; 38(10):e116. PMC: 2879533. DOI: 10.1093/nar/gkq072. View

Li H, Ruan J, Durbin R . Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 2008; 18(11):1851-8. PMC: 2577856. DOI: 10.1101/gr.078212.108. View

Shen Y, Wan Z, Coarfa C, Drabek R, Chen L, Ostrowski E . A SNP discovery method to assess variant allele probability from next-generation resequencing data. Genome Res. 2009; 20(2):273-80. PMC: 2813483. DOI: 10.1101/gr.096388.109. View

10.

Abdulla M, Ahmed I, Assawamakin A, Bhak J, Brahmachari S, Calacal G . Mapping human genetic diversity in Asia. Science. 2009; 326(5959):1541-5. DOI: 10.1126/science.1177074. View

11.

Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14):1754-60. PMC: 2705234. DOI: 10.1093/bioinformatics/btp324. View

12.

Langmead B, Schatz M, Lin J, Pop M, Salzberg S . Searching for SNPs with cloud computing. Genome Biol. 2009; 10(11):R134. PMC: 3091327. DOI: 10.1186/gb-2009-10-11-r134. View

13.

McKernan K, Peckham H, Costa G, McLaughlin S, Fu Y, Tsung E . Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding. Genome Res. 2009; 19(9):1527-41. PMC: 2752135. DOI: 10.1101/gr.091868.109. View

14.

Yi X, Liang Y, Huerta-Sanchez E, Jin X, Cuo Z, Pool J . Sequencing of 50 human exomes reveals adaptation to high altitude. Science. 2010; 329(5987):75-8. PMC: 3711608. DOI: 10.1126/science.1190371. View

15.

Wheeler D, Srinivasan M, Egholm M, Shen Y, Chen L, McGuire A . The complete genome of an individual by massively parallel DNA sequencing. Nature. 2008; 452(7189):872-6. DOI: 10.1038/nature06884. View

16.

Lee W, Jiang Z, Liu J, Haverty P, Guan Y, Stinson J . The mutation spectrum revealed by paired genome sequences from a lung cancer patient. Nature. 2010; 465(7297):473-7. DOI: 10.1038/nature09004. View

17.

Langmead B, Trapnell C, Pop M, Salzberg S . Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3):R25. PMC: 2690996. DOI: 10.1186/gb-2009-10-3-r25. View

18.

Abecasis G, Altshuler D, Auton A, Brooks L, Durbin R, Gibbs R . A map of human genome variation from population-scale sequencing. Nature. 2010; 467(7319):1061-73. PMC: 3042601. DOI: 10.1038/nature09534. View

19.

Conrad D, Keebler J, DePristo M, Lindsay S, Zhang Y, Casals F . Variation in genome-wide mutation rates within and between human families. Nat Genet. 2011; 43(7):712-4. PMC: 3322360. DOI: 10.1038/ng.862. View

20.

Ng S, Turner E, Robertson P, Flygare S, Bigham A, Lee C . Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 2009; 461(7261):272-6. PMC: 2844771. DOI: 10.1038/nature08250. View