title: Next-generation sequencing of human opioid receptor genes based on a custom AmpliSeq™ library and ion torrent personal genome machine.
abstract: BACKGROUND
  The opioid system is involved in the control of pain, reward, addictive behaviors and vegetative effects. Opioids exert their pharmacological actions through the agonistic binding at opioid receptors and variation in the coding genes has been found to modulate opioid receptor expression or signaling. However, a limited selection of functional opioid receptor variants is perceived as insufficient in providing a genetic diagnosis of clinical phenotypes and therefore, unrestricted access to opioid receptor genetics is required.
  
  
  METHODS
  Next-generation sequencing (NGS) workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes related to the opioid receptor group (OPRM1, OPRD1, OPRK1, SIGMA1, OPRL1) on an Ion PGM™ Sequencer. A cohort of 79 previously studied chronic pain patients was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding. In-silico analysis was performed using SNP and Variation Suite® software.
  
  
  RESULTS
  The amplicons covered approximately 90% of the target sequence. A median of 2.54×106reads per run was obtained generating a total of 35,447 nucleotide reads from each DNA sample. This identified approximately 100 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19, including functional variants such as the OPRM1 rs1799971 SNP (118 A>G) as the most scientifically regarded variant or rs563649 SNP coding for μ-opioid receptor splice variants. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.
  
  
  CONCLUSIONS
  Results suggested that the NGS approach based on AmpliSeq™ libraries and Ion PGM sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of opioid receptor genes. The method includes the variants studied so far for functional associations and adds a large amount of genetic information as a basis for complete analysis of human opioid receptor genetics and its functional consequences.
authors.affiliation: Institute of Clinical Pharmacology, Goethe - University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.
authors.forename: Dario
authors.initials: D
authors.lastname: Kringel
authors.affiliation: Institute of Clinical Pharmacology, Goethe - University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology TMP, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address: j.loetsch@em.uni-frankfurt.de.
authors.forename: Jörn
authors.initials: J
authors.lastname: Lötsch
mesh_terms: D015723:Gene Library; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D011957:Receptors, Opioid / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D011957:Receptors, Opioid
keywords: Addiction; Analgesia; Molecular diagnosis; Mutations; NGS sequencing; Opioid system