Table 1 Comparison of basic characteristics associated with selected techniques for studying active microbial populations in situ that can be combined with shotgun sequencing

From: Capturing the genetic makeup of the active microbiome in situ

Cell process targeted

Technique

Advantages

Disadvantages

Biases/assumptions

Reference

DNA synthesis

BrdU labeling (B, S)

Can be used as single-cell tool if combined with fluorescent antibody staining

Low labeling efficiency

Rate of uptake varies by cell; may be toxic to some cells

Borneman, 1999; Yin et al., 2000*

 

DNA-SIP (B)

Link of metabolic function to identity

Long incubation time; cross-feeding problem; contamination prone; high GC DNA problematic; dependent on commercially available labeled compoundsa

Cell division may be stimulated by substrate addition and not reflect in situ growth rates

Radajewski et al., 2000*

 

Peak-to-trough ratios in metagenomes (S+B), iRep

No sample incubation necessary; samples can be frozen immediately

Reliant on database; affected by community complexity.

 

Korem et al., 2015;* Brown et al., 2016*

Transcription

RNA-SIP (B)

High phylogenetic resolution; link of metabolic function to taxonomic identity

Dependent on commercially available labeled compounds; cross-feeding potentialb

Cell division may be stimulated by substrate addition and not reflect in situ growth rates

Radajewski et al., 2003*

 

Metatranscriptomics (S+B)

Samples can be frozen immediately; inexpensive; fast

mRNA abundance not necessarily indicative of protein levels and enzymatic activities; high transcriptome coverage necessary; modulation may occur during sampling

RNA abundance does not directly correlate with cell activity

Leininger et al., 2006

Amino-acid biosynthesis

BONCAT (+FISH/FACS) (S+B)

No known effect on growth and translation activity

High amino acid conc. in sample diminishes/suppresses signal

Cell growth may be stimulated by added amino acids; dependence on uptake mechanism

Hatzenpichler et al., 2014; Hatzenpichler et al., 2016*

Lipid, carbohydrate, amino-acid biosynthesis

D2O+Raman microspectrometry (S)

D2O concentration required not known to be toxic to any cells; no stimulation effect expected in hydrated samples; can be combined with FISH; stimulation experiments with unlabeled substrates possible

Low-throughput so far

Different physiologies lead to different incorporation rates of deuterium (for example, autotrophs vs heterotrophs)

Berry et al., 2015*

Bacterial reduction potential

Redox sensor green+FACS (S+B)

Rapid response to redox activity within cell; stable fluorescence signal; quick cell penetration; no detrimental effect on core cell functions known; stimulation experiments with unlabeled substrates possible

Incubated cells cannot be frozen; not all microbial species may transport redox sensor green equally well into their cells

 

Kalyuzhnaya et al., 2008*

  1. Techniques are further characterized as allowing for single-cell approaches (S) and/or bulk community analysis (B). References given denote those of either first use of the respective method or the first combined use with (any) sequence data (marked with an asterisk).
  2. Abbreviations: BONCAT, bioorthogonal non-canonical amino-acid tagging; BrdU, bromodeoxyuridine; FACS, fluorescence-activated cell sorting; FISH, fluorescence in situ hybridization; iRep, index of replication; SIP, stable isotope probing.
  3. aDNA-SIP may be used with 18O-H2O, which—to our knowledge—has not been used with metagenomics or single-cell sequencing.
  4. bRNA-SIP may be used with 18O-H2O, which—to our knowledge—has not been used with metagenomics or single-cell sequencing.
Back to article page