TY - CHAP
T1 - Whole cell immunomagnetic enrichment of environmental microbial consortia using rRNA-Targeted Magneto-FISH
AU - Trembath-Reichert, Elizabeth
AU - Green-Saxena, Abigail
AU - Orphan, Victoria J.
N1 - Funding Information:
We acknowledge Annelie (Pernthaler) Wendeberg, Rachel Poretsky, Joshua Steele for their contributions towards the development and optimization of Magneto-FISH. Stephanie Cannon, Jen Glass, Kat Dawson, Hiroyuki Imachi, and Caltech Genomics Center are also acknowledged for their assistance with various aspects of this project. Funding for this work was provided by the Gordon and Betty Moore foundation and a DOE early career grant (to V. J. O.) and NIH/NRSA training grant 5 T32 GM07616 (to E. T. R.).
PY - 2013
Y1 - 2013
N2 - Magneto-FISH, in combination with metagenomic techniques, explores the middle ground between single-cell analysis and complex community characterization in bulk samples to better understand microbial partnerships and their roles in ecosystems. The Magneto-FISH method combines the selectivity of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with immunomagnetic capture to provide targeted molecular and metagenomic analysis of co-associated microorganisms in the environment. This method was originally developed by Pernthaler et al. (Pernthaler et al., 2008; Pernthaler & Orphan, 2010). It led to the discovery of new bacterial groups associated with anaerobic methane-oxidizing (ANME-2) archaea in methane seeps, as well as provided insight into their physiological potential using metagenomics. Here, we demonstrate the utility of this method for capturing aggregated consortia using a series of nested oligonucleotide probes of differing specificity designed to target either the ANME archaea or their Deltaproteobacteria partner, combined with 16S rRNA and mcrA analysis. This chapter outlines a modified Magneto-FISH protocol for large- and small-volume samples and evaluates the strengths and limitations of this method predominantly focusing on (1) the relationship between FISH probe specificity and sample selectivity, (2) means of improving DNA yield from paraformaldehyde-fixed samples, and (3) suggestions for adapting the Magneto-FISH method for other microbial systems, including potential for single-cell recovery.
AB - Magneto-FISH, in combination with metagenomic techniques, explores the middle ground between single-cell analysis and complex community characterization in bulk samples to better understand microbial partnerships and their roles in ecosystems. The Magneto-FISH method combines the selectivity of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with immunomagnetic capture to provide targeted molecular and metagenomic analysis of co-associated microorganisms in the environment. This method was originally developed by Pernthaler et al. (Pernthaler et al., 2008; Pernthaler & Orphan, 2010). It led to the discovery of new bacterial groups associated with anaerobic methane-oxidizing (ANME-2) archaea in methane seeps, as well as provided insight into their physiological potential using metagenomics. Here, we demonstrate the utility of this method for capturing aggregated consortia using a series of nested oligonucleotide probes of differing specificity designed to target either the ANME archaea or their Deltaproteobacteria partner, combined with 16S rRNA and mcrA analysis. This chapter outlines a modified Magneto-FISH protocol for large- and small-volume samples and evaluates the strengths and limitations of this method predominantly focusing on (1) the relationship between FISH probe specificity and sample selectivity, (2) means of improving DNA yield from paraformaldehyde-fixed samples, and (3) suggestions for adapting the Magneto-FISH method for other microbial systems, including potential for single-cell recovery.
KW - ANME
KW - CARD-FISH
KW - Cell separation
KW - Metagenomics
KW - Microbial association
KW - Symbiosis
KW - Syntrophy
UR - http://www.scopus.com/inward/record.url?scp=84884575093&partnerID=8YFLogxK
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U2 - 10.1016/B978-0-12-407863-5.00002-2
DO - 10.1016/B978-0-12-407863-5.00002-2
M3 - Chapter
C2 - 24060114
AN - SCOPUS:84884575093
SN - 9780124078635
T3 - Methods in Enzymology
SP - 21
EP - 44
BT - Microbial Metagenomics, Metatranscriptomics, and Metaproteomics
PB - Academic Press Inc
ER -