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A simple method for gene phasing using mate pair sequencing

Kendall W Cradic1, Stephen J Murphy2, Travis M Drucker3, Robert A Sikkink4, Norman L Eberhardt56, Claudia Neuhauser7, George Vasmatzis2* and Stefan KG Grebe1*

Author Affiliations

1 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA

2 Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA

3 Information Technology, Mayo Clinic, Rochester, MN 55905, USA

4 Adavanced Genomics Technology Center, Mayo Clinic, Rochester, MN 55905, USA

5 Department of Medicine, Division of Endocrinology, Mayo Clinic, Rochester, MN 55905, USA

6 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA

7 Biomedical Informatics and Computational Biology, University of Minnesota Rochester, 111 South Broadway, Suite 300, Rochester, MN 55904, USA

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BMC Medical Genetics 2014, 15:19  doi:10.1186/1471-2350-15-19

Published: 6 February 2014



Recessive genes cause disease when both copies are affected by mutant loci. Resolving the cis/trans relationship of variations has been an important problem both for researchers, and increasingly, clinicians. Of particular concern are patients who have two heterozygous disease-causing mutations and could be diagnosed as affected (one mutation on each allele) or as phenotypically normal (both mutations on the same allele). Several methods are currently used to phase genes, however due to cost, complexity and/or low sensitivity they are not suitable for clinical purposes.


Long-range amplification was used to select and enrich the target gene (CYP21A2) followed by modified mate-pair sequencing. Fragments that mapped coincidently to two heterozygous sites were identified and used for statistical analysis.


Probabilities for cis/trans relationships between heterozygous positions were calculated along with 99% confidence intervals over the entire length of our 10 kb amplicons. The quality of phasing was closely related to the depth of coverage and the number of erroneous reads. Most of the error was found to have been introduced by recombination in the PCR reaction.


We have developed a simple method utilizing massively parallel sequencing that is capable of resolving two alleles containing multiple heterozygous positions. This method stands out among other phasing tools because it provides quantitative results allowing confident haplotype calls.

Gene phasing; Compound heterozygosity; Haplotype; Next generation sequencing