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Resolving genomic disorder–associated breakpoints within segmental DNA duplications using massively parallel sequencing

Nature Protocols volume 9, pages 1496–1513 (2014)Cite this article

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Abstract

The most common recurrent copy-number variants associated with autism, developmental delay and epilepsy are flanked by segmental duplications. Complete genetic characterization of these events is challenging because their breakpoints often occur within high-identity, copy-number polymorphic paralogous sequences that cannot be specifically assayed using hybridization-based methods. Here we provide a protocol for breakpoint resolution with sequence-level precision. Massively parallel sequencing is performed on libraries generated from haplotype-resolved chromosomes, genomic DNA or molecular inversion probe (MIP)-captured breakpoint-informative regions harboring paralog-distinguishing variants. Quantification of sequencing depth over informative sites enables breakpoint localization, typically within several kilobases to tens of kilobases. Depending on the approach used, the sequencing platform, and the accuracy and completeness of the reference genome sequence, this protocol takes from a few days to several months to complete. Once established for a specific genomic disorder, it is possible to process thousands of DNA samples within as little as 3–4 weeks.

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Figure 1: General workflow for breakpoint resolution.
Figure 2: Sequencing-based breakpoint resolution strategy.
Figure 3: Resolution of 17q21.31 microdeletion breakpoints using WGS data.
Figure 4: Resolution of NAHR-associated RHD duplication and deletion breakpoints by MIP capture and sequencing.

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Acknowledgements

We thank J. Huddleston for assistance in testing analysis software and preparing it for public access; P. Sudmant for analyzing the number of SUNKs in windows across the reference genome; and T. Brown for assistance with manuscript preparation. X.N. is supported by a National Science Foundation Graduate Research Fellowship under grant no. DGE-1256082. This work was supported by US National Institutes of Health grants HG004120 and HG002385 to E.E.E. E.E.E. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington, USA

    Xander Nuttle, Andy Itsara, Jay Shendure & Evan E Eichler

  2. Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington, USA

    Evan E Eichler

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  1. Xander Nuttle
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  4. Evan E Eichler
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Contributions

X.N., A.I., J.S. and E.E.E. developed the protocol. X.N. and E.E.E. wrote the paper, with input and approval from all coauthors.

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Correspondence to Evan E Eichler.

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Competing interests

E.E.E. is on the scientific advisory board (SAB) of DNAnexus and was an SAB member of Pacific Biosciences (2009–2013) and SynapDx (2011–2013). The remaining authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Primers used for MIP experiments. (XLSX 33 kb)

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Nuttle, X., Itsara, A., Shendure, J. et al. Resolving genomic disorder–associated breakpoints within segmental DNA duplications using massively parallel sequencing. Nat Protoc 9, 1496–1513 (2014). https://doi.org/10.1038/nprot.2014.096

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