Unravel the hidden protistan diversity: application of blocking primers to suppress PCR amplification of metazoan DNA

Applied Microbiology and Biotechnology
Shangjin Tan, Hongbin Liu

Abstract

Planktonic protists, including both autotroph and heterotroph, have been recognized as a major contributor to primary production and consumers of bacteria, archaea and picophytoplankton. However, the understanding of protistan diversity is typically impeded by the large amount of metazoans when employing universal primers to environmental samples. In this study, we developed 3 blocking primers to inhibit the amplification of metazoan DNA in PCR. First, we optimized the design and concentration of blocking primers by using 2 metazoans and 3 protists in quantitative PCR. No significant difference (q > 0.05) was found in protistan community structure at phylum, family or OTU levels and genetic diversity between samples amplified with and without adding blocking primers, indicating that the blocking primer does not alter the composition of protistan community. An application to samples with a high abundance of metazoans showed that the blocking primer can reduce 42.1-72.4% of metazoan sequences, resulting the retrieval of a higher protistan richness and diversity. The use of the blocking primer helped to identify protistan community composition from a large size fraction, which is usually dominated by metazoans. Protistan community...Continue Reading

References

Feb 2, 2000·Applied and Environmental Microbiology·F von WintzingerodeU B Göbel
Nov 27, 2002·Antonie van Leeuwenhoek·Jens Boenigk, Hartmut Arndt
Jan 11, 2003·Proceedings of the National Academy of Sciences of the United States of America·Purificación López-GarcíaDavid Moreira
Feb 23, 2005·Molecular Ecology·L E Blankenship, A A Yayanos
May 10, 2006·Applied Microbiology and Biotechnology·Shantanu Karkare, Deepak Bhatnagar
May 16, 2008·Biometrical Journal. Biometrische Zeitschrift·Torsten HothornPeter Westfall
Jun 20, 2008·FEMS Microbiology Reviews·Daniel VaulotHervé Moreau
Aug 12, 2008·Nature·Mikhail V Zubkov, Glen A Tarran
Mar 26, 2009·Molecular Ecology·Bruce E DeagleSimon N Jarman
Oct 16, 2009·Trends in Microbiology·Rebecca J GastDavid A Caron
Jun 7, 2011·Annual Review of Microbiology·Ramon Massana
Jun 28, 2011·Bioinformatics·Robert C EdgarRob Knight
Dec 8, 2011·Molecular Ecology Resources·R O'RorkeA Jeffs
Feb 22, 2012·Applied and Environmental Microbiology·Yun-Chi LinAlexandra Z Worden
Mar 10, 2012·The ISME Journal·William OrsiVirginia Edgcomb
Mar 31, 2012·Annual Review of Marine Science·David A CaronAstrid Schnetzer
Aug 1, 2012·Nature Methods·Diego DarribaDavid Posada
Oct 11, 2012·PloS One·Ryuji J Machida, Nancy Knowlton
Jan 19, 2013·Molecular Biology and Evolution·Kazutaka Katoh, Daron M Standley
May 16, 2013·The Journal of Eukaryotic Microbiology·David A Caron
Nov 13, 2014·Frontiers in Microbiology·Darren J ParrisFrank J Stewart
May 23, 2015·Science·Colomban de VargasEric Karsenti
Oct 10, 2015·The ISME Journal·Massimo C PerniceRamon Massana

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Datasets Mentioned

BETA
PRJNA384701

Methods Mentioned

BETA
electrophoresis
PCR
in vitro transcription
Assay
PCRs
light microscopy

Software Mentioned

Mothur
R
jModelTest
MAFFT
robust
multcomp
PhyML

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