title: Examining changes in bacterial abundance in complex communities using next-generation sequencing is enhanced with quantitative PCR.
abstract: Changes in the composition of microbial communities are often examined using high-throughput sequencing (HTS). Here we analyzed bar-coded Illumina sequencing data from a previous work describing the microbial community found in the sputum of a cystic fibrosis patient by itself or in combination with qPCR to measure the absolute abundance of Pseudomonas aeruginosa and Burkholderia multivorans. Through this comparison we were able to determine the computational analysis accuracy of sequencing data to measure the relative abundance of specific taxa. While no correlation was found between relative abundance and absolute abundance of P. aeruginosa or B. multivorans, we did find conclusions derived from HTS data alone differed from those derived from a combination of HTS and qPCR. Our results highlight the importance of using qPCR with HTS when characterizing organisms in microbial communities having a dominant taxon.
authors.affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, 28223, USA.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Stokell
authors.affiliation: Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, NC, 28223, USA.|PerkinElmer, Inc., 940 Winter Street, Waltham, MA, 02451, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Hamp
authors.affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, 28223, USA. trsteck@uncc.edu.
authors.forename: Todd R
authors.initials: TR
authors.lastname: Steck
mesh_terms: D058491:Bacterial Load; D015373:Bacterial Typing Techniques / Q000379:methods*; D044822:Biodiversity; D019117:Burkholderia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D003550:Cystic Fibrosis / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D064307:Microbiota; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013183:Sputum / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: Cystic fibrosis; High-throughput sequencing; Microbial communities; qPCR