Microbiome Sample Preparation – Tips and Tricks 

September 2020

From the human gut to varied environments such as Antarctic soil, ocean water, or acidic hot spring biofilms, microbiome can be found in diverse conditions. Regardless of the origin of the sample, microbiome researchers require high yields and unbiased results to analyze microbial communities. But researchers often face complex and difficult-to-lyse samples. Furthermore, inhibitors present in these samples and background DNA and RNA can cause inaccurate quantification of nucleic acids and may inhibit downstream applications.

In May 2020, we hosted a webinar, in which tips and tricks for the preparation and purification of challenging microbiome samples were discussed. We captured a lot of interesting questions from our webinar attendees. In this blog, we present our compilation of the top 15 questions on the topic of sample preparation, along with insightful answers from our QIAGEN microbiome experts: Dr. Doerte Lehmann, Scientist Microbiome Product Development and Julie Sean, Global Product Manager Microbiome Sample Preparation products.

Q&A from Tips and Tricks Webinar

How to minimize the variability and contamination during collection of the sample?

We recommend to stabilize the soil or stool sample using the RNAprotect Tissue Reagent (cat.no. 76104, 76106) and its supplementary microbiome stabilization protocol.

Reducing the risks of contaminating your sample depends on multiple factors. Contaminating your sample while collecting it is one component. However, following good working practices while isolating the DNA is probably more important. If you’re careful when collecting your sample (for example, keeping exposure to a minimum) you don’t need any protection such as masks and gloves. The small amount of your microbiome that might end up in the collection tube will most likely not affect the sequencing. But if you want to be sure not to contaminate the environment you can wear gloves and masks. However, if you’re collecting from a dangerous environment you should definitely wear gloves, masks, and other appropriate PPE while sampling.

Do you have any recommendation for inhibitor removal from microbiome samples?

At QIAGEN, all the Power kits contain the patented Inhibitor Removal Technology® (IRT). It was especially designed for the removal of PCR-inhibitors from soil, stool, water, air or even biofilm samples. The latest generation of kits have “Pro” in their names, they are equipped with an upgraded version of IRT, performed in one step instead of two in the legacy kits. You will find it in the QIAamp PowerFecal Pro DNA Kit (cat.no. 51804) or in the DNeasy PowerSoil Pro Kits (cat.no. 47014, 47016). For higher throughput, you can use the DNeasy 96 PowerSoil Pro QIAcube HT Kit (cat.no. 47021) for automated sample purification. For a manual high-throughput protocol, use the DNeasy 96 PowerSoil Pro Kit (cat.no. 47017).

How to adjust the lysis protocol when interested in fungi?

Usually you would have to adjust the lysis but the DNeasy PowerSoil Pro Kit is already developed to extract DNA from bacteria as well as fungi.

Is there any difference when extracting DNA from mice stool as compared to human stool?

No, nothing needs to be adjusted, you can extract DNA from mice stool as you would from human stool. Mouse pellets can be very dry, if you are finding low liquid recovery (<650ul) after beating in CD1, you can add up to 200 ul PBS to the pellets prior to bead beating.

How to adjust sample preparation when working with high saline samples?

We’ve seen higher DNA yields with saline soil when we washed the soil with PBS before extracting DNA. To increase yields from saline soil, wash the soil with sterile PBS as follows:

  1. Mix 0.25 grams of the soil sample with 1 ml sterile PBS in a collection tube.
  2. Invert a few times to mix.
  3. Centrifuge at 10,000 x g for 2 minutes to pellet all the cells and solid material. Discard the supernatant.
  4. Repeat the above washing procedure.

If needed, washes can be repeated.

What is the minimum amount of sample material required for saliva?

We usually use swabs to collect saliva from a patient. This swab is then processed like a stool or soil sample. To process saliva directly, 250 µl of saliva is added in place of the sample.

What are the common inhibitors in tropical soil/peat soil samples?

Tropical soil and peat soil is usually high in humic acids. The inhibitor removal step in the DNeasy PowerSoil Pro Kit is optimized to remove humic acid.

How to remove the inhibitors from FFPE samples?

Usually FFPE samples don’t contain high amounts of inhibitors. The challenge with FFPE samples is the deparaffinization as well as the crosslink-removal. Both are essential. The QIAamp DNA FFPE Advanced Kit (cat.no 56604) would be a good option. If you are still concerned that the DNA contains inhibitors, these inhibitors can be removed using DNeasy PowerClean Pro Cleanup Kit (cat.no. 12997-50).

What modifications to DNA extraction protocols can be used to reduce bias, particularly when targeting both bacteria and archaea?

The DNeasy PowerSoil Pro Kit (cat.no. 47014, 47016) for soil samples and the QIAamp PowerFecal Pro DNA Kit (cat.no. 51804) for stool samples also extract DNA from archaea.

We have seen a much better representation of archaea in 16S sequencing when using the DNeasy Power Soil Pro Kit compared to the DNeasy PowerSoil Kit.

How to obtain usable DNA from decayed samples?

A group of researchers analyzed mastics found at a Mesolithic site, Huseby Klev, in western Sweden1. Mastics are chewing “gums” made from tree pitch or other natural substances chewed both recreationally and for use in tool making around the world. Of eight mastics processed, three of them provided usable human DNA. They noted that with QIAGEN’s QIAamp PowerFecal DNA Kit, now QIAamp PowerFecal Pro DNA Kit (cat.no. 51804). To learn more, read our blog.

Is there a specific kit for DNA extraction in tissue?

This solution wasn’t tested in our R&D but one possibility is to combine two kits. The idea is to start with the disruption of the tissue using the ballcone beads from the QIAamp Fast DNA Tissue Kit (cat.no. 51404), and to proceed with the DNA extraction using the QIAamp DNA Microbiome Kit (cat.no. 51704).

You should keep in mind that the ballcone bead will most likely lyse some of the bacteria (the ballcone is not very efficient in lysing bacteria but it will lyse some). The subsequent step (degrading host DNA) will then not only degrade host DNA but also degrade the DNA of the lysed bacteria.

If you’re doing 16S sequencing you don’t need host depletion and could try QIAamp PowerFecal Pro DNA in combination with a ballcone (needed for tissues high in cartilage) or not depending on your type of tissue.

What are the options if DNA yield is too low (less than 1 ng)?

You could extract DNA from multiple aliquots of your sample and combine them after DNA isolation. You could switch to a different DNA extraction method. The DNeasy PowerSoil Pro Kit obtains very high DNA yields from the most challenging samples.

It is not recommended to use less than 1 ng DNA as input level for your library preparation. Such low levels would introduce taxonomic biases that would result in a definitive misrepresentation of the microbiome.

How can I detect inhibitors in my samples?

After you obtain a visually clear sample, measure the absorbance ratios at 260/280 nm and 260/230 nm. Ratios between 1.8 and 2.0 indicate a high purity for the DNA extracted.

Is it possible to profile bacterial communities using <1 ng/µl gDNA?

A study from 20182 performed a systematic and comprehensive assessment of the impact of various experimental parameters on the resulting 16S rRNA gene library and showed that microbial community representation can be affected by gDNA concentration and 16S rRNA gene yield. On the basis of these results, we recommend a minimum concentration above 4 x 10-2 ng/µl gDNA input, and, ideally, >2 x 10-1 ng/µl, to achieve an unbiased representation of the microbial community. The use of gDNA input levels of ≤1.6 x 10-3 ng/µl is not recommended because such levels would introduce taxonomic biases that would result in a definitive misrepresentation of the microbiome.

How to obtain longer DNA fragments from extraction?

To obtain longer DNA fragments you can use the MagAttract HMW DNA Kit (cat.no. 67563) (average length: 100 -200 kB) or Genomic Tips (average length: 50 -100 kb). For the lysis bead beating should be avoided. Enzymatical lysis of the microbial sample is recommended.

We hope the above information was useful to you and are always happy to continue answering your questions. Feel free to visit our microbiome webpage to learn more about the QIAGEN take on microbiome research, or contact us directly.

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Inga Irle image

Inga Irle

Dr. Inga Irle, is a Senior Strategic Marketing manager based in QIAGEN’s Hilden office, Germany. She obtained her Master of Science degree in Biochemistry in 2006 focusing on molecular medicine and biomarker discovery and received her PhD in 2009 working in the field of cancer and epigenetics. Dr. Irle joined QIAGEN in 2010 and was the Global Product Manager for various products, including qPCR and Epigenetics. She is currently responsible for the global marketing activities around QIAGEN’s Microbiome products and Life Science Sample preparation instruments.

References:

  1. Kashuba N, Kirdök E, Damlien H, Manninen MA, Nordqvist B, Persson P, Götherström A. Ancient DNA from mastics solidifies connection between material culture and genetics of mesolithic hunter-gatherers in Scandinavia. Communication Biology. 2019 May 15;2:185.
  2. Multinue et. al (2018) Systematic Bias Introduced by Genomic DNA Template Dilution in 16S rRNA Gene-Targeted Microbiota Profiling in Human Stool Homogenates, mSphere
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