Probe-based negative selection for underrepresented phylotypes in large environmental clone libraries

Journal of Microbiological Methods
Jenni HultmanLars Paulin

Abstract

Studies based on cloning and sequencing to investigate microbial diversity in a vast range of samples has become widespread in recent years. Results have revealed immense microbial diversity in many different environments, but also dominance of a few sequence types in the constructed clone libraries. Here we describe a method to enrich the clone libraries by avoiding sequencing of known, abundant sequence types, instead focusing on novel, rare ones. The protocol is based on gridding the PCR products from clone libraries on membranes and hybridisation of species-specific probes. Clones that do not give positive hybridisation results are sequenced. This method was used for fungal clone libraries from compost samples. Altogether 1536 clones were gridded and six probes used. From these clones, 59% hybridised with a probe, and therefore, only 41% of the clones were sequenced. In addition, 384 samples were sequenced to verify the hybridisation results. The numbers of false-negative (5.2%) and false-positive (3.9%) hybridisations were low. This method provides a mean of lowering the costs of sequencing projects and speeding up the process of characterising microbial diversity in environmental samples. The method is especially suitable...Continue Reading

References

Oct 5, 1990·Journal of Molecular Biology·S F AltschulD J Lipman
Feb 6, 1998·FEMS Microbiology Reviews·F von WintzingerodeE Stackebrandt
Feb 12, 2002·Bioinformatics·James K BonfieldRodger Staden
Mar 16, 2002·Science·Philippe VandenkoornhuyseJ Peter W Young
Jun 29, 2002·Applied and Environmental Microbiology·Lea ValinskyJames Borneman
Nov 27, 2002·Applied and Environmental Microbiology·Lea ValinskyJames Borneman
Jun 5, 2003·Journal of Microbiological Methods·Veljo Kisand, Johan Wikner
Sep 6, 2003·Science·Christopher W SchadtSteven K Schmidt
Apr 7, 2004·Science·J Craig VenterHamilton O Smith
Mar 23, 2004·Nucleic Acids Research·Robert C Edgar
Jul 30, 2004·Nature·Silvia G AcinasMartin F Polz
Apr 16, 2005·Science·Paul B EckburgDavid A Relman
Apr 23, 2005·Science·Susannah Green TringeEdward M Rubin
Jul 8, 2005·Applied and Environmental Microbiology·Noah FiererRobert B Jackson
Aug 27, 2005·Science·T P Curtis, W T Sloan
Aug 30, 2005·Clinica Chimica Acta; International Journal of Clinical Chemistry·Alexander Loy, Levente Bodrossy
Sep 10, 2005·Applied and Environmental Microbiology·Heath E O'BrienRytas Vilgalys
Feb 8, 2006·Applied and Environmental Microbiology·Karin NeubertStefan G R Wirsel
Aug 2, 2006·Proceedings of the National Academy of Sciences of the United States of America·Mitchell L SoginGerhard J Herndl
Oct 6, 2007·Science·Julie A HuberMitchell L Sogin
Nov 6, 2007·Applied and Environmental Microbiology·M PitkärantaH Rintala

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Citations

Jun 11, 2011·Environmental Science and Pollution Research International·Sari KauppiAki Sinkkonen
Jan 1, 2009·BMC Microbiology·Jenni HultmanPetri Auvinen

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