2.1.1. Extraction of bacterial community DNA
Extraction is the first step towards identifying the microbial communities present in a sample (Fig. 1). Extraction methods for diverse bacterial communities must include a method to disrupt the cell walls of spores and the more recalcitrant bacteria (Marmur, 1961). We recommend using a method that includes bead-beating because that is one of the best methods for cell disruption, and it has been the method of choice for recent bee-associated NGS surveys (Mattila et al., 2012; McFrederick et al., 2012; Moran et al., 2012).
- First determine what you consider to be your sample: the entire gut or part of the gut. It is very important to work in a clean, sterile environment using sterile materials and tools. Unless you intend to include the microbial community occurring on the insect cuticle, it is imperative to disinfect and clean the surface of the bees before dissections. Microbes and their nucleic acids can be removed from bee cuticle using a 1% aqueous solution of chlorine (using either sodium dichloro-s-triazinetrione-dihydrate (swimming pool or spa chlorine) or sodium hyperchlorite (bleach)). Soak each bee for at least 2 min but not more than 7 min., then rinse three times in sterile, purified water. This can be done in a 24 well plate on a shaker table, or in a series of 1.5 ml disposable centrifuge tubes with gentle mixing on a vortex mixer. It is important that all of the chlorine be removed from the bees prior to dissection because chlorine degrades DNA and thus can inhibit the PCR reactions.
- Once you obtain your sample, place it in a sterile 2 ml microcentrifuge tube with a sterile 5 mm stainless steel bead (QIAGEN, Valencia, CA), 500 μl of 0.1 mm glass beads (Scientific Industries, Inc.;Bohemia, NY, USA), and 500 μl RLT buffer (QIAGEN; Valencia, CA, USA) with 10 μl of β-mercaptoethanol per ml buffer. Note that β-mercaptoethanol (including the used buffer) must be handled as hazardous waste.
- Run in tissue lyser or bead beater at 30 Hz for 5 min.
- Centrifuge samples briefly to separate the beads and the buffer. If a foam layer has developed, it can be eliminated with a longer centrifugation period (several minutes at 5000 rpm at 5°C).
- In a new tube add 100 μl of 100% ethanol and 100 μl of supernatant (200 μl of 100% ethanol and 200 μl of supernatant may be used for small samples, but it is important to treat all samples within a study the same so that the data are comparable). Gently mix using a vortex mixer. Centrifuge briefly, if needed, to consolidate the sample, which may get spread around in the tube.
- For DNA recovery, apply the sample from step 5 to a QIAamp mini spin column and follow the tissue protocol of QIAamp DNA minikit (QIAGEN, Valencia, CA) (starting at the step where the supernatant is applied to the spin column, Step 7 on page 35 in the handbook published 4/2010).
- Elute DNA in 30 μl of sterilized nanopure water, or in the provided QIAGEN buffer AE if long-term storage at -20 is desired.
- Quantify DNA and adjust to a standard concentration across all samples. We have used 20 ng/μl, but higher concentrations may be desirable with small samples.
Fig. 1. Suggested protocol for data collection, processing, and analysis of bacterial community data using 16S rRNA next generation sequencing (NGS).