Immune Homeostasis pp 175-187

Part of the Methods in Molecular Biology book series (MIMB, volume 979)

Designs for Massively Parallel Sequencing Approaches to Identify Causal Mutations in Human Immune Disorders



Massively parallel sequencing technologies provide new opportunities to discover causal variants and ­narrow down candidate genes responsible for human Mendelian disorders. Such information can in turn provide new insights into understanding the basic science behind, as well as improving diagnosis and treatment for, these disorders. In this chapter, we review experimental design and data analysis for sequencing studies of human immune disorders. We discuss optimal experimental designs for sample selection and sequencing approaches, as well as key aspects of data analysis such as filtering and prioritization of identified variants.

Key words

Mendelian diseases Genetic Inheritance Patterns Exome sequencing Whole genome sequencing Causal variants Filtering and prioritization of variants 


  1. 1.
    Bolze A et al (2010) Whole-exome-sequencing-based discovery of human FADD deficiency. Am J Hum Genet 87:873–881PubMedCrossRefGoogle Scholar
  2. 2.
    Ng SB et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272–276PubMedCrossRefGoogle Scholar
  3. 3.
    Liu L et al (2011) Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis. J Exp Med 208:1635–1648PubMedCrossRefGoogle Scholar
  4. 4.
    Li FY et al (2011) Second messenger role for Mg2+ revealed by human T-cell immunodeficiency. Nature 475:471–476PubMedCrossRefGoogle Scholar
  5. 5.
    O’Roak BJ et al (2011) Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet 43:585–589PubMedCrossRefGoogle Scholar
  6. 6.
    Johnson JO et al (2010) Exome sequencing reveals VCP mutations as a cause of familial ALS. Neuron 68:857–864PubMedCrossRefGoogle Scholar
  7. 7.
    Wang JL et al (2010) TGM6 identified as a novel causative gene of spinocerebellar ataxias using exome sequencing. Brain 133:3510–3518PubMedCrossRefGoogle Scholar
  8. 8.
    Musunuru K et al (2010) Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med 363:2220–2227PubMedCrossRefGoogle Scholar
  9. 9.
    Krawitz PM et al (2010) Identity-by-descent filtering of exome sequence data identifies PIGV mutations in hyperphosphatasia mental retardation syndrome. Nat Genet 42:827–829PubMedCrossRefGoogle Scholar
  10. 10.
    Ng SB et al (2010) Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 42:30–35PubMedCrossRefGoogle Scholar
  11. 11.
    Lalonde E et al (2010) Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing. Hum Mutat 31:918–923PubMedCrossRefGoogle Scholar
  12. 12.
    Pierce SB et al (2010) Mutations in the DBP-deficiency protein HSD17B4 cause ovarian dysgenesis, hearing loss, and ataxia of Perrault Syndrome. Am J Hum Genet 87:282–288PubMedCrossRefGoogle Scholar
  13. 13.
    Hoischen A et al (2010) De novo mutations of SETBP1 cause Schinzel-Giedion syndrome. Nat Genet 42:483–485PubMedCrossRefGoogle Scholar
  14. 14.
    Gilissen C et al (2010) Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet 87:418–423PubMedCrossRefGoogle Scholar
  15. 15.
    Ng SB et al (2010) Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet 42:790–793PubMedCrossRefGoogle Scholar
  16. 16.
    Botstein D, Risch N (2003) Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease. Nat Genet 33(Suppl):228–237PubMedCrossRefGoogle Scholar
  17. 17.
    Choi M et al (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA 106:19096–19101PubMedCrossRefGoogle Scholar
  18. 18.
    Ku CS et al (2011) Revisiting Mendelian disorders through exome sequencing. Hum Genet 129:351–370PubMedCrossRefGoogle Scholar
  19. 19.
    Okou DT et al (2007) Microarray-based genomic selection for high-throughput resequencing. Nat Methods 4:907–909PubMedCrossRefGoogle Scholar
  20. 20.
    Gnirke A et al (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27:182–189PubMedCrossRefGoogle Scholar
  21. 21.
    Bainbridge MN et al (2010) Whole exome capture in solution with 3 Gbp of data. Genome Biol 11:R62PubMedCrossRefGoogle Scholar
  22. 22.
    Sulonen AM et al (2011) Comparison of solution-based exome capture methods for next generation sequencing. Genome Biol 12:R94PubMedCrossRefGoogle Scholar
  23. 23.
    Brownstein Z et al (2011) Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in middle eastern families. Genome Biol 12:R89PubMedCrossRefGoogle Scholar
  24. 24.
    Nikopoulos K et al (2010) Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy. Am J Hum Genet 86:240–247PubMedCrossRefGoogle Scholar
  25. 25.
    Rehman AU et al (2010) Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79. Am J Hum Genet 86:378–388PubMedCrossRefGoogle Scholar
  26. 26.
    Tewhey R et al (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27:1025–1031PubMedCrossRefGoogle Scholar
  27. 27.
    Johansson H et al (2011) Targeted resequencing of candidate genes using selector probes. Nucleic Acids Res 39:e8PubMedCrossRefGoogle Scholar
  28. 28.
    Chepelev I et al (2009) Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq. Nucleic Acids Res 37:e106PubMedCrossRefGoogle Scholar
  29. 29.
    Cirulli ET et al (2010) Screening the human exome: a comparison of whole genome and whole transcriptome sequencing. Genome Biol 11:R57PubMedCrossRefGoogle Scholar
  30. 30.
    Lupski JR et al (2010) Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy. N Engl J Med 362:1181–1191PubMedCrossRefGoogle Scholar
  31. 31.
    Horner DS et al (2010) Bioinformatics approaches for genomics and post genomics applications of next-generation sequencing. Brief Bioinform 11:181–197PubMedCrossRefGoogle Scholar
  32. 32.
    Meyerson M et al (2010) Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet 11:685–696PubMedCrossRefGoogle Scholar
  33. 33.
    Bentley DR et al (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53–59PubMedCrossRefGoogle Scholar
  34. 34.
    Sherry ST et al (2001) dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29:308–311PubMedCrossRefGoogle Scholar
  35. 35.
    Consortium, T. G. P (2010) A map of human genome variation from population-scale sequencing. Nature 467:1061–1073CrossRefGoogle Scholar
  36. 36.
    Altshuler DM et al (2010) Integrating common and rare genetic variation in diverse human populations. Nature 467:52–58PubMedCrossRefGoogle Scholar
  37. 37.
    Stitziel NO et al (2011) Computational and statistical approaches to analyzing variants identified by exome sequencing. Genome Biol 12:227PubMedCrossRefGoogle Scholar
  38. 38.
    Adzhubei IA et al (2010) A method and server for predicting damaging missense mutations. Nat Methods 7:248–249PubMedCrossRefGoogle Scholar
  39. 39.
    Cooper GM et al (2010) Single-nucleotide evolutionary constraint scores highlight disease-causing mutations. Nat Methods 7:250–251PubMedCrossRefGoogle Scholar
  40. 40.
    Ng PC, Henikoff S (2003) SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res 31:3812–3814PubMedCrossRefGoogle Scholar
  41. 41.
    Pollard KS et al (2010) Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res 20:110–121PubMedCrossRefGoogle Scholar
  42. 42.
    Siepel A et al (2005) Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res 15:1034–1050PubMedCrossRefGoogle Scholar
  43. 43.
    Asthana S et al (2007) Analysis of sequence conservation at nucleotide resolution. PLoS Comput Biol 3:e254PubMedCrossRefGoogle Scholar
  44. 44.
    Mailman MD et al (2007) The NCBI dbGaP database of genotypes and phenotypes. Nat Genet 39:1181–1186PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Human Immunological Diseases Unit, Laboratory of Host DefensesNational Institute of Allergy and Infectious Diseases, National Institutes of HealthBethesdaUSA

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