Microbiome Services

Services Available

DNA Extraction (pending)
Sample Preparation  
Sequencing
Analysis

16S Microbiome Workflow

For standard analyses, Vantage uses the 16S V4 amplification and sequencing approach developed at the Schloss lab.  The full protocol can be seen here: https://github.com/SchlossLab/MiSeq_WetLab_SOP/blob/master/MiSeq_WetLab_SOP_v4.md


Microbiome sample preparation generally encompases the following steps:

  1. DNA extraction/purification (link to SOP for DNA extraction using bead-beating)

  2. Inclusion of controls:

    1. Water blanks (negative controls)

    2. Mock communities (for assessment of sequencing accuracy, optional)

  3. Sample validation using generic 16S primers (optional, but highly recommended)

    1. PCR using 27f, 1492r (or others at your discretion)

    2. Validation of amplification of all samples using gel electrophoresis (~1.5 kb band)

  4. Dual-barcoded amplification of V4 16S region (link to paper?)

    1. PCR using dual barcoded primers (including water blanks, and mock community)

    2. Validation of amplification using gel electrophoresis (~250 bp band)

  5. PCR cleanup/normalization of DNA concentration of all samples

    1. Final target ~2-3 ug/uL in 20 uL (double check)

  6. Pooling of all samples for sequencing

  7. Sequencing

    1. MiSeq, 2X250 bp for V4

  8. Analysis

    1. Mothur (Vantage)/Qiime


Sample Suggestions and Requirements

If this is your first microbiome experiment, some thoughts on design of experiments:
http://www.sciencedirect.com/science/article/pii/S0092867414008642

In short, it’s complicated, and you need to be sure you are aware of your question, the number and cost of all of your samples, and have a null hypothesis to attempt to discard.  

Current conventional wisdom is the use of 10 samples per condition.  For mouse/rodent studies, it is likely that you will need to consider the co-housing/littermate effects on the microbiome.

  1. Raw Samples:

    1. Currently, Vantage is working on obtaining equipment for DNA extraction of microbiome samples.  At that time, we will be able to accept unextracted samples.  Until then, you can look at our “Best Practices” section for DNA extraction suggestions.

  2. DNA for library preparation and sequencing

    1. Vantage can accept extracted DNA, arrayed in 96-well plates.  

    2. It is suggested that DNA be validated by use of simple amplification of 16S via PCR, and gel electrophoresis.  Procedures and reagents can be found here: (to procedures FAQ, 16S amplicon testing)

    3. For PCR prep and sample analysis, each sample must be quantified for DNA concentration before PCR (either by user, or Vantage staff)

  3. User made libraries

    1. Vantage cannot accept responsibility for poorly sequenced libraries, or read-distribution for user-made libraries

    2. Samples must be Illumina compatible, barcoded, pooled, and ready to sequence.

    3. This can all be accomplished following the same protocol used by Vantage scientists

    4. Protocol:

      1. You can follow this protocol: https://github.com/SchlossLab/MiSeq_WetLab_SOP/blob/master/MiSeq_WetLab_SOP_v4.md

      2. Supplies suggested are

    5. Supplies:

      1. Vantage has stocks of the V4 dual-barcoded primers used in our protocol available for purchase. (less than $10/96 samples)

      2. Vantage is able to provide normalization plates and reagents at $40/96 well plate

      3. Vantage does not carry stocks of the standard Pfx mastermix for this SOP (http://www.thermofisher.com/order/catalog/product/A1051001)


Analysis

Analysis of 16S rRNA gene abundance is a method to identify members of individual communities which are shared between samples of a particular type, and excluded from another.  This can either be binary (presence/absence), or a difference in relative abundance.  In all cases, this analysis requires significant computational resources, and some degree of expertise.  Analysis performed at Vantage will make use of the Mothur software resources (www.mothur.org) to look for reliable markers.  This work will be based around the standard analysis workflow, visible here: https://github.com/scholzmb/VantageMothur  which is adapted from the MiSeq tutorial found at mothur.org.  



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