TITLE:
Evaluation of Molecular Techniques in Characterization of Deep Terrestrial Biosphere
AUTHORS:
Malin Bomberg, Mari Nyyssönen, Aura Nousiainen, Jenni Hultman, Lars Paulin, Petri Auvinen, Merja Itävaara
KEYWORDS:
DGGE, 16S rRNA Gene, Amplicon Library, Sequencing, Deep Subsurface
JOURNAL NAME:
Open Journal of Ecology,
Vol.4 No.8,
May
30,
2014
ABSTRACT:
A suite of
molecular methods targeting 16S rRNA genes (i.e.,
DGGE, clone and high-throughput [HTP] amplicon library sequencing) was used to
profile the microbial communities in deep Fennoscandian crystalline bedrock
fracture fluids. Variation among bacterial 16S rRNA genes was examined with two
commonly used primer pairs: P1/P2 and U968f/U1401r. DGGE using U968f/ U1401r
mostly detected β-, γ-proteobacteria and Firmicutes, while
P1/P2 primers additionally detected other proteobacterial clades and candidate
divisions. However, in combination with clone libraries the U968f/U1401r
primers detected a higher bacterial diversity than DGGE alone. HTP amplicon
sequencing with P1/P2 revealed an abundance of the DGGE bacterial groups as
well as many other bacterial taxa likely representing minor components of these
communities. Archaeal diversity was investigated via DGGE or HTP amplicon sequencingusing primers A344F/ 519RP. The majority of archaea detected with HTP amplicon sequencing
belonged to uncultured Thermoplasmatales and Pendant 33/DHVE3, 4, 6 groups.
DGGE of the same samples detected mostly SAGMEG and Methanosarcinales archaea,
but almost none of those were revealed by HTP amplicon sequencing. Overall, our
results show that the inferred diversity and composition of microbial
communities in deep fracture fluids is highly dependent on analytical technique
and that the method should be carefully selected with this in mind.