Abstract
Sequencing taxonomic marker genes is a powerful tool to interrogate the composition of microbial communities. For example, bacterial and fungal community composition can be evaluated in parallel using the 16S ribosomal RNA gene for bacteria or the internal transcribed spacer region in fungi. These are conserved regions that are universal to a taxonomic clade, yet have undergone some degree of evolution such that different lineages can be differentiated. Conserved regions are used for design of universal priming sites that allow amplification of the marker gene out of a mixed microbial community. Here, we describe our standard operating procedure to collect and sequence 16S rRNA and ITS1 amplicons from human skin. We use the 16S rRNA V1-V3 region for skin samples, as it has greater power for classifying common staphylococci in the skin. This protocol is adapted for 454 pyrosequencing of amplicons.
Keywords: Microbiome, 16S rRNA, ITS, Skin microbiome extraction
Materials and Reagents
Equipment
Procedure
Representative data
Acknowledgments
This protocol is based on the data generated and analyses performed in Oh et al. (2013). Skin sampling procedures were designed by Dr. Heidi Kong, and DNA extraction and amplification procedures by Clayton Deming, Cynthia Ng, and Elizabeth Grice at the National Institutes of Health.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
The yeast cell lysis buffer is the yeast cell lysis solution in the MasterPure kit, yes. And yes, then there is no need to use the lysis buffer from the PureLink kit.
Hi Ella,Try the high-sensitivity (not broad range) Qubit kit. We find that NanoDrop is not accurate at low concentrations. Yield varies very significantly by skin site. For example, for the inner nostril, you might expect 50 ng/uL; for a low biomass sites like the forearm or inner elbow you might achieve 0.5 ng/uL. Run your negative control in your PCR (also include a negative control for the PCR only), and see if you get a detectable band with yield--that will be informative as to potential contamination in your collection method.
Hi Julia, Thanks a lot for your prompt reply. This is very helpful. I will try PCR next.
Dear Julia, I tried to run a PCR using 2ul of the microbiome elute (isolated from scalp swab and air control) and V1_27F and V3_534R primers. I also included a water control in this PCR reaction. I only saw a very faint band around 8kb in the scalp sample and both negative controls. Given that the band size is large( I was expecting a smaller size) and the faint signal of the band, I think this band might be just a background band. I am wondering if you normally see a distinct band when you do the 16S rRNA PCR. The PCR was done using the program provided in 16S amplification section in your protocol. I think the problem for my experiment is that I didn't break up the cells enough to release DNA. According to the protocol, it looks like we are only using the PureLink genomic DNA mini kit for DNA purification (binding DNA to the column and washes with two buffers). The actual cell lysis was done using the MasterPure kit. Since this kit is designed for yeast DNA extraction, do you think it can affect the efficiency on extracting bacterial DNA? I think I will try to lyse the cells longer in the bead beater and see if I can improve the lysis. I will also really appreciate it if you have any suggestions and tips on isolating skin microbiome that I can try in my next experiment. thanks a lot for you help.
Hi Ella,We've had good experience co-extracting bacterial DNA using this protocol (we use the same eluate for both 16S and ITS sequencing). Depending on the skin site, we may or may not see a band on the gel. High yield sites (e.g., inner nares) can have a band. Did you do a positive control? You might run a positive extraction control, like the nares. In our experience, the combination of the lysis buffer + bead beating is sufficient to lyse both bacterial and fungal cells.
Hi Julia, Thanks a lot for your response. My samples were collected from scalp, so I guess this may not be one of the high yield sites. Thanks for the suggestion of using the inner nares as a positive control. We thought about running a positive but didn't know better about what would be a good control. Thanks again for helping me out. I really appreciate it.
Yes, this is the yeast cell lysis solution from the MasterPure Kit.
Thank you for the prompt reply.Just to be sure: after step B3:"Short spin tubes, then open and add 1 μl of ReadyLyse and 250 μl of Yeast Cell Lysis Solution."we will have 350 μl of Yeast Cell Lysis Solution in our tube. Why are we only starting with 100 μl for the extraction? 100 μl is rather small volume to moisten the Catch All swab. I would normally use 1000 μl of the SCF-1 solution (described in the methodology of the HMP for sampling skin).Could you shortly explain the rationale here?
Because of the low biomass in the skin, we try to keep the volumes low. This amount is adequate to moisten the swab for sampling a 4cm2 area. If you are planning on sampling a larger area, it'd be worth experimenting with larger volumes.