title: A simple method for gene phasing using mate pair sequencing.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Kendall W
authors: KW
authors: Cradic
authors: Stephen J
authors: SJ
authors: Murphy
authors: Travis M
authors: TM
authors: Drucker
authors: Robert A
authors: RA
authors: Sikkink
authors: Norman L
authors: NL
authors: Eberhardt
authors: Claudia
authors: C
authors: Neuhauser
authors: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA. vasmatzis.george@mayo.edu.
authors: George
authors: G
authors: Vasmatzis
authors: Stefan K G
authors: SK
authors: Grebe
mesh_terms: D006239:Haplotypes / Q000235:genetics*; D006579:Heterozygote; D016133:Polymerase Chain Reaction; D011336:Probability; D012107:Research Design; D017421:Sequence Analysis / Q000379:methods*; D013255:Steroid 21-Hydroxylase / Q000235:genetics
chemical_list: D013255:Steroid 21-Hydroxylase