Microbiome Best Practices

Table of Contents:


Sample considerations

Mouse Studies: Cage effects

In multiple papers, studies have indicated that littermates, and co-housed mice will have similar microbiomes, along with the more “well known” issues with consistent rack/light/temperature effects on outcomes.  There are many suggested solutions to this, depending on opportunities to co-house animals of different treatments, or sharing bedding between cages.  Two potential references for this are here:

References:

Macpherson, A. J., & McCoy, K. D. (2015). Standardised animal models of host microbial mutualism. Mucosal Immunology, 8(3), 476–86. 

Goodrich, J. K., Di Rienzi, S. C., Poole, A. C., Koren, O., Walters, W. A., Caporaso, J. G., … Ley, R. E. (2014). Conducting a microbiome study. Cell, 158(2), 250–62. http://doi.org/10.1016/j.cell.2014.06.037


DNA extraction:

There have been multiple studies to determine the effects of different DNA extraction methods on the subsequent amplification and analysis of microbiome samples.  From these analyses, the winner is the MoBio Powersoil kit (https://mobio.com/powersoil-dna-isolation-kit.html), with no, or minor modifications (Lu et al. 2015, Mackenzie et al. 2015).  As an example of modifications:

From Lu et al. (2015), looking at porcine gut/fecal extractions:

POW method

DNA extraction with POW was performed according to the manufacturer’s instructions with modifications of bead beating time and additional pre-elution incubation. Sample aliquot was added into the lysing matrix with lysis buffer supplied. Lysing matrix was then blended with mini Bead Beater (BioSpec, Bartlesville, US) at 4,800 oscillations per minute for 60 seconds. The remaining steps (including protein removal and DNA washing) were performed as recommended. DNA was also eluted with 50 μL RNAnase-free water with pre-elution incubation at 50°C for 5 minutes.

References:

Lu, Y., Hugenholtz, P., & Batstone, D. J. (2015). Evaluating DNA Extraction Methods for Community Profiling of Pig Hindgut Microbial Community. PloS One, 10(11), e0142720.

Wagner Mackenzie, B., Waite, D. W., & Taylor, M. W. (2015). Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences. Frontiers in Microbiology, 6, 130. 



Sample Validation using generic 16S amplification:

We recommend investigators verify that their DNA extracts contain sufficient DNA with few enough PCR inhibitors to reliably amplify 16S rRNA genes.  Our current recommendation is to amplify each sample using generic 27F and 1492R, using the following PCR protocol:

 

Primers:

27F - AGRGTTTGATYMTGGCTCAG
1492R - GGYTACCTTGTTACGACT


PCR set up:

Component: ul/reaction (20 ul reactions)
5X buffer 4
MgCl2 1.2
dNTPs 0.2
Taq 0.2
Forward primer 1.0
Reverse primer 1.0
Water 11.4
DNA template 1.0

 

PCR Steps:

  1. 2 min @ 95°C
  2. 30 cycle of:
    1. 30sec @ 95°C
    2. 30sec @ 55°C
    3. 1:30 @ 72°C
  3. 10 min @ 72°C
  4. Keep at 4°C


Once completed, load each sample onto a gel, with appropriate size ladder (expected band is 1.5 kb), and validate that all samples are able to properly amplify 16S rRNA genes.  This step is NOT performed at Vantage.  If this has not been performed by investigators, pre-drop-off, costs for any samples that cannot be amplified properly will still be charged to your account in the case of PCR failure, unless this step has been performed.



PCR cleanup/DNA concentration normalization

Once PCR using the dual barcoded primers is completed, there are 2 required steps before the amplicons can be sequenced:

  1. PCR cleanup (removal of extraneous DNA, primers, enzymes, etc)

  2. DNA concentration normalization between samples


The MiSeq SOP (above) recommends use of the Sequal prep (http://products.invitrogen.com/ivgn/product/A1051001) kit for a combined PCR cleanup/DNA concentration normalization.  This is our recommendation as well.  However, the kit is only available in a 10X pack of 96 well plates (total of 960 samples) for ~$550, making the cost prohibitive.  We are currently working out pricing to stock and sell individual plates.  The only alternative method (also outlined in the SOP), is gel-based amplicon cleanup, and manual DNA concentration/normalization.  

For additional questions, please contact Matthew Scholz.  E: matthew.b.scholz@vanderbilt.edu  P: 615-875-9503