title: Preferential Amplification of Pathogenic Sequences.
abstract: The application of next generation sequencing (NGS) technology in the diagnosis of human pathogens is hindered by the fact that pathogenic sequences, especially viral, are often scarce in human clinical specimens. This known disproportion leads to the requirement of subsequent deep sequencing and extensive bioinformatics analysis. Here we report a method we called "Preferential Amplification of Pathogenic Sequences (PATHseq)" that can be used to greatly enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and 10-mer oligonucleotides called "non-human primers" that do not match the most abundant human transcripts, but instead selectively match transcripts of human pathogens. Instead of using random primers in the construction of cDNA libraries, the PATHseq method recruits these short non-human primers, which in turn, preferentially amplifies non-human, presumably pathogenic sequences. Using this method, we were able to enrich pathogenic sequences up to 200-fold in the final sequencing library. This method does not require prior knowledge of the pathogen or assumption of the infection; therefore, it provides a fast and sequence-independent approach for detection and identification of human viruses and other pathogens. The PATHseq method, coupled with NGS technology, can be broadly used in identification of known human pathogens and discovery of new pathogens.
authors.affiliation: Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Ge
authors.affiliation: 1] Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA [2] Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, USA.
authors.forename: Jayme
authors.initials: J
authors.lastname: Parker
authors.affiliation: Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA.
authors.forename: Sang
authors.initials: S
authors.lastname: Chul Choi
authors.affiliation: Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Layer
authors.affiliation: Alaska State Public Health Laboratories, Anchorage, Alaska, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Ross
authors.affiliation: Alaska State Public Health Laboratories, Anchorage, Alaska, USA.
authors.forename: Bernard
authors.initials: B
authors.lastname: Jilly
authors.affiliation: 1] Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA [2] Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, USA.
authors.forename: Jack
authors.initials: J
authors.lastname: Chen
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D019288:Herpesvirus 8, Human / Q000235:genetics*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010222:Parainfluenza Virus 1, Human / Q000235:genetics*; D014777:Virus Diseases / Q000175:diagnosis / Q000235:genetics / Q000401:mortality
chemical_list: D004279:DNA, Viral