- What are my options for examining the microbiome?
- Can I look at any sample type?
- What controls should I consider?
- What primer sets do you have for targeted sequencing?
- Do you offer microbial culture as well?
- Single-end or paired-end sequencing for the microbiome?
- How much will it cost?
- How long will it take?
- How do you handle data analysis?
- So I can just give you my samples then?
What are my options for examining the microbiome?
We use either targeted sequencing of ‘marker genes’, such as the 16S rRNA gene, or ‘shotgun’ metagenomics. Several factors, including experimental goals, timeframe, and available $$ will influence which method you choose. Marker gene sequencing is relatively inexpensive, can accommodate large numbers of samples (in the hundreds), but takes us some time to turn around and ultimately does not give species- or strain-level resolution of the community. In contrast, metagenomics is more expensive, is typically lower throughput, but we can turn this around much faster and this method can provide a higher resolution view of the community, including functional potential (i.e. gene content).
Can I look at any sample type?
Sure, but there are some considerations. Typically, we handle samples with relatively high microbial biomass (e.g. stool or soil). If you are interested in profiling the skin microbiome, or environmental surfaces, then you have to consider that you’ll be dealing with extremely low biomass. This means your study design should include extra precautions to control for contamination and artifacts.
What controls should I consider?
All runs should include the following controls:
- Water Blank - this is just water from the kit used to extract the DNA. This controls for any potential nucleic acid contamination present in your kit reagent. This ‘kitome’ has been well documented in this paper to be an important issue.
- Air Blank - not essential with high biomass samples.
- Mock communities - These are simple community mixtures of known bacteria made in our lab.
|Bacterium||ATCC Strain Designation||MockA||MockB||MockC|
Streptococcus equi ssp equi
Salmonella typhii strain 1490
Mycobacterium avium subspecies paratuberculosis
Staphylococcus schleiferi strain 2317-03
What primer sets do you have for targeted sequencing?
We have the V4 16S primers described by Pat Schloss’ lab here with enough barcodes to multiplex X samples.
Do you offer microbial culture as well?
Not really. We offer basic culture for common aerobic or facultative anaerobic organisms like Staphylococcus, Streptococcus, and E. coli. For more involved culture-dependent assays, we work closely with the PennVet Clinical Microbiology Laboratory and Elliot Freedman at the PennCHOP Microbial Culture Core
Single-end or paired-end sequencing for the microbiome?
For marker gene sequencing it is not only important that you do paired-end sequencing, but the read length must be such that the paired reads overlap completely (usually 250 bp paired-end). This ensures that you have the highest possible consensus sequence across the amplicon. Single-end or only partially overlapping paired-end could result in higher degrees of uncorrected sequence error that will appear as true sequence diversity (complicating your determination of community composition). You can read more about this here.
For shotgun metagenomics, paired-end is ideal.
How much will it cost?
The exact costs will depend on your particular experiment, however, here are some back-of-the-envelope numbers for shotgun metagenomics sequencing to help you budget (all at Tier 3):
- DNA extraction is $15/sample
- Library prep is $30/sample
- Sequencing on our NextSeq 2000 machine is ~$6600 for a paired-end 150 bp P3 run. This will generate 1.2 billion read-pairs, which is a sufficient depth of coverage for about 100 samples.
How long will it take?
Our turn-around time varies, but we typically take 2-4 weeks to go from samples to sequences. Analysis time depends on your question and interests.
How do you handle data analysis?
We offer many options for analysis and, ultimately, we will choose an analysis method based on your question, timeline and interest in learning some of the analysis methods yourself. For rapid turnaround and pathogen detection from shotgun metagenomics data, we have partnered with CosmosID and can deliver data to you within minutes of completing sequencing for $80/sample. If this commercial service is not essential for you, or you want a more in-depth analysis of your data, we use Curtis Huttenhower’s BioBakery suite of tools or Sunbeam as a pipeline for analysis of metagenomic data. We use QIIME2 and Mothur for the analysis of marker gene sequencing data. Finally, we also developed MicrobiomeDB for comparing your data to large publically available studies.
In addition to the SOPs provided on this site, we have also put together a number of detailed posts on the lab blog that walk you through these types of analyses. Assistance is also available through 1:1 consultations with CHMI staff.
So I can just give you my samples then?
Our tiered service model affords flexibility in how we work with you. See the table below for a summary of the tiers. If you are interested in using our reagents, we recommend starting with Tier 2 as you are learning the protocol, then moving to Tier 1 for future projects once you are comfortable.
- Tier 1 – I just need the reagents; I can do the bench work myself
- Tier 2 – I need reagents and some hands-on help.
- Tier 3 – I need reagents and would like you to do all the bench work