title: DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies.
abstract: The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden.
authors.forename: Luisa W
authors.initials: LW
authors.lastname: Hugerth
authors.affiliation: Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Science for Life Laboratory, Stockholm, Sweden.
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Wefer
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden.
authors.forename: Sverker
authors.initials: S
authors.lastname: Lundin
authors.affiliation: Department of Preparedness, Swedish Institute for Communicable Disease Control, Stockholm, Sweden Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Hedvig E
authors.initials: HE
authors.lastname: Jakobsson
authors.affiliation: Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Science for Life Laboratory, Stockholm, Sweden.
authors.forename: Mathilda
authors.initials: M
authors.lastname: Lindberg
authors.affiliation: Department of Preparedness, Swedish Institute for Communicable Disease Control, Stockholm, Sweden Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sandra
authors.initials: S
authors.lastname: Rodin
authors.affiliation: Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Science for Life Laboratory, Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Engstrand
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden anders.andersson@scilifelab.se.
authors.forename: Anders F
authors.initials: AF
authors.lastname: Andersson
mesh_terms: D000465:Algorithms; D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D039301:Computers, Molecular; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012417:Rumen / Q000382:microbiology; D012623:Seawater / Q000382:microbiology; D012984:Software
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S