title: Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB.
abstract: We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.
authors.forename: Guohong
authors.initials: G
authors.lastname: Liu
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.|Singlera Genomics, Inc., La Jolla, California, 92037, United States of America.
authors.forename: Christopher Q
authors.initials: CQ
authors.lastname: Weston
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.
authors.forename: Long K
authors.initials: LK
authors.lastname: Pham
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.|Singlera Genomics, Inc., La Jolla, California, 92037, United States of America.|San Diego State University, San Diego, California, United States of America.
authors.forename: Shannon
authors.initials: S
authors.lastname: Waltz
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.
authors.forename: Helen
authors.initials: H
authors.lastname: Barnes
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.|Singlera Genomics, Inc., La Jolla, California, 92037, United States of America.
authors.forename: Paula
authors.initials: P
authors.lastname: King
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.
authors.forename: Dan
authors.initials: D
authors.lastname: Sphar
authors.affiliation: Zova Systems, LLC, San Diego, California, 92129, United States of America.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Yamamoto
authors.affiliation: FLIR Systems, Inc., La Jolla, California, 92037, United States of America.|Singlera Genomics, Inc., La Jolla, California, 92037, United States of America.|San Diego State University, San Diego, California, United States of America.
authors.forename: R Allyn
authors.initials: RA
authors.lastname: Forsyth
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D019003:Deoxyribonuclease HpaII / Q000302:isolation & purification / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism; D015723:Gene Library; D005827:Genetics, Microbial / Q000379:methods*; D023281:Genomics / Q000379:methods*; D006801:Humans; D054892:Metagenome; D064307:Microbiota / Q000235:genetics; D017422:Sequence Analysis, DNA; D013183:Sputum / Q000382:microbiology; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal; D018744:DNA, Plant; D016054:DNA, Protozoan; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D044503:5-Methylcytosine; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII; C059678:mcrB protein, E coli