title: The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms.
abstract: BACKGROUND
  Molecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.
  
  
  RESULTS
  We generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.
  
  
  CONCLUSIONS
  Recent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.
authors.affiliation: Department of Biology, Duke University, Durham, NC 27708, USA. peter.larsen@duke.edu.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Larsen
authors.forename: Amy M
authors.initials: AM
authors.lastname: Heilman
authors.forename: Anne D
authors.initials: AD
authors.lastname: Yoder
mesh_terms: D000818:Animals; D001483:Base Sequence; D016565:Cheirogaleidae / Q000235:genetics*; D016000:Cluster Analysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D011110:Polymorphism, Genetic; D018035:Receptors, Odorant / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D019147:Vomeronasal Organ / Q000502:physiology
chemical_list: D018035:Receptors, Odorant