title: Intra-genomic variation in G + C content and its implications for DNA stable isotope probing.
abstract: Combining deoxyribonucleic acid (DNA-based) stable isotope probing (DNA-SIP) with high-throughput sequencing provides a powerful culture-independent means to link microbial metabolic function to genomic information and taxonomic identity. DNA buoyant density (BD) in isopycnic gradients is dependent on both isotope incorporation and G + C content. G + C content varies across a genome but is constrained at rrn operons; hence, the ability to resolve isotopically labelled DNA from unlabelled DNA in SIP may vary between small subunit-ribosomal nucleic acid (SSU rRNA) amplicon and shotgun-read sequencing applications. We tested this hypothesis by evaluating the G + C content of genomic DNA fragments that encompassed either an SSU rRNA template ('amplicon-fragments') or a shotgun read template ('shotgun-fragments'). We find that, contrary to expectations, the BD distribution of amplicon-fragments is non-normal and can be highly skewed. Furthermore, the BD distribution of amplicon-fragments can differ substantially from that of shotgun-fragments from the same genome. Our findings demonstrate the impact of G + C content on the downstream applications of DNA-SIP, which will aid in proper experimental design and the development of statistical tests to accurately identify sequences derived from isotopically labelled DNA.
authors.affiliation: Department of Crop and Soil Sciences, Cornell University, 306 Tower Road, Ithaca, NY, 14853, USA.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Youngblut
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Buckley
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D001482:Base Composition; D002247:Carbon Isotopes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D023281:Genomics; D009587:Nitrogen Isotopes / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D004269:DNA, Bacterial; D009587:Nitrogen Isotopes