title: Characterization and identification of hidden rare variants in the human genome.
abstract: BACKGROUND
  By examining the genotype calls generated by the 1000 Genomes Project we discovered that the human reference genome GRCh37 contains almost 20,000 loci in which the reference allele has never been observed in healthy individuals and around 70,000 loci in which it has been observed only in the heterozygous state.
  
  
  RESULTS
  We show that a large fraction of this rare reference allele (RRA) loci belongs to coding, functional and regulatory elements of the genome and could be linked to rare Mendelian disorders as well as cancer. We also demonstrate that classical germline and somatic variant calling tools are not capable to recognize the rare allele when present in these loci. To overcome such limitations, we developed a novel tool, named RAREVATOR, that is able to identify and call the rare allele in these genomic positions. By using a small cancer dataset we compared our tool with two state-of-the-art callers and we found that RAREVATOR identified more than 1,500 germline and 22 somatic RRA variants missed by the two methods and which belong to significantly mutated pathways.
  
  
  CONCLUSIONS
  These results show that, to date, the investigation of around 100,000 loci of the human genome has been missed by re-sequencing experiments based on the GRCh37 assembly and that our tool can fill the gap left by other methods. Moreover, the investigation of the latest version of the human reference genome, GRCh38, showed that although the GRC corrected almost all insertions and a small part of SNVs and deletions, a large number of functionally relevant RRAs still remain unchanged. For this reason, also future resequencing experiments, based on GRCh38, will benefit from RAREVATOR analysis results. RAREVATOR is freely available at http://sourceforge.net/projects/rarevator .
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. albertomagi@gmail.com.
authors.forename: Alberto
authors.initials: A
authors.lastname: Magi
authors.affiliation: Laboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics and Institute of Clinical Physiology, National Research Council, Pisa, Italy. romina.daurizio@gmail.com.
authors.forename: Romina
authors.initials: R
authors.lastname: D'Aurizio
authors.affiliation: Medical Genetics Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy. palombo.flavia@gmail.com.
authors.forename: Flavia
authors.initials: F
authors.lastname: Palombo
authors.affiliation: Institute for Biomedical Technologies, National Research Council, Milan, Italy. ingrid.cifola@itb.cnr.it.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Cifola
authors.affiliation: Department of Neuroscience, Pharmacology and Child Health, University of Florence, Florence, Italy. lorenzotattini@gmail.com.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Tattini
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. robybass88@gmail.com.
authors.forename: Roberto
authors.initials: R
authors.lastname: Semeraro
authors.affiliation: Medical Genetics Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy. tommaso.pippucci@gmail.com.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Pippucci
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. betti.giusti@unifi.it.
authors.forename: Betti
authors.initials: B
authors.lastname: Giusti
authors.affiliation: Medical Genetics Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy. egf.giovanni.romeo@gmail.com.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Romeo
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. rosanna.abbate@unifi.it.
authors.forename: Rosanna
authors.initials: R
authors.lastname: Abbate
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. gfgensini@gmail.com.
authors.forename: Gian Franco
authors.initials: GF
authors.lastname: Gensini
mesh_terms: D000483:Alleles; D030541:Databases, Genetic; D056426:Genetic Loci; D014644:Genetic Variation / Q000235:genetics*; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D020407:Internet; D009369:Neoplasms / Q000235:genetics / Q000473:pathology; D020641:Polymorphism, Single Nucleotide; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D017422:Sequence Analysis, DNA; D014584:User-Computer Interface