title: High-resolution melt analysis for rapid comparison of bacterial community compositions.
abstract: In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.
authors.forename: Mathis Hjort
authors.initials: MH
authors.lastname: Hjelmsø
authors.forename: Lars Hestbjerg
authors.initials: LH
authors.lastname: Hansen
authors.forename: Jacob
authors.initials: J
authors.lastname: Baelum
authors.forename: Louise
authors.initials: L
authors.lastname: Feld
authors.forename: William E
authors.initials: WE
authors.lastname: Holben
authors.forename: Carsten Suhr
authors.initials: CS
authors.lastname: Jacobsen
mesh_terms: D001419:Bacteria / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D044822:Biodiversity; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D017753:Ecosystem; D005308:Fertilizers / Q000032:analysis; D057166:High-Throughput Screening Assays / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000235:genetics; D012988:Soil Microbiology; D044366:Transition Temperature
chemical_list: D004269:DNA, Bacterial; D005308:Fertilizers; D012336:RNA, Ribosomal, 16S