Friday 11 November 2016

Sampling, Storing and Extracting DNA

The way in which DNA from a sample is handled and extracted from cells can introduce biases into the analysis of a microbial community. How it's done will differ depending on whether you're sampling from soil, ocean water, or faeces. I'm going to concentrate on sampling from gut contents and faeces.

Taking the sample

Working with chickens is far easier than working with people. They don't have the same social rules about a researcher waiting eagerly for them to produce a faecal sample. People tend to like to do their pooing in private, which adds an additional challenge to collecting samples. Person or poultry, the basic question remains the same: "How can I get this microbial community into a pot without contaminating it with the bacteria that are covering everything?"

There will be bacteria present in the air, on our hands and  our clothes so we need to consider how best to minimise these contaminants. The majority of studies I've read about the chicken intestinal microbiome don't give many details about how they took their samples. Some groups have removed the gastrointestinal tract, transferred them to individual sterile bags and kept them on ice until contents can be sampled in a cleaner environment such as a laboratory (1-4)⁠, but they don't give much more detail than that. 

A microbiome sequencing gut sample kit
Tony Webster from San Francisco, California, UBiome - Microbiome Sequencing Gut Bacteria Sample Kit (17238556660)CC BY 2.0
I don't think it's too much to imagine that you're a surgeon. During surgery, everything has to be as clean as possible, you, your equipment, your work surfaces. This is all to prevent bacterial contamination of your patient, so why shouldn't we extend the same to our samples? Sometimes we need to be even more careful, since even if you kill bacteria, although they can't grow, their DNA can still contribute to the sample. There was a good Reddit post dealing with this in the context of sterilising medical implants, but the general idea is the same.

When taking or handling samples, we can minimise contamination by using aseptic protocols. These include measures such as:
  • Wearing personal protective equipment (gowns, gloves, hair nets, face masks, etc.) to minimise contamination from skin, clothing and hair.
  • Using 70% ethanol to clean work benches and surfaces.
  • Working within a fume hood which can be treated with UV light between samples.
An example of personal protective equipment in a laboratory
Photo: U.S. Food and Drug Administration/Public Domain
The UV light goes back to what I was saying earlier about dead bacteria still being able to contribute DNA to samples. UV light damages and breaks down DNA, so it'll help to minimise this contamination. You can also soak any equipment you're going to reuse in 30% bleach for 30 minutes.

There are lots of ways to prevent contamination once the sample has got to the laboratory. If you want to read about them in more detail, there's a good link here about aseptic techniques.

Fresh Frozen Faeces

If DNA extraction can’t be carried out on fresh samples, then samples will have to be stored to stop damage to the DNA. The recommended method is direct freezing at -80°C (5)⁠. This doesn't affect the viability of the DNA, and you'll get similar results between fresh and frozen samples (6). There might actually be an advantage to freezing your samples. The ratio of Firmicutes to Bacteroides has been observed to change after sample freezing, with an increase in DNA from Firmicutes. The suspected cause is that freezing samples improves DNA extraction from gram-positive bacteria (7)⁠. However, this hasn't been replicated in all studies, so there's probably a variety of factors at play. There's even some information that suggests you can store faecal samples at room temperature for up to two weeks, and it won't significantly affect the bacteria that you find in the sample (8). Personally, I would reason that the faster you can extract the DNA the better, and if you can't extract the DNA it's probably better to freeze the sample just to be sure. 

If you can't beat them...

Now that we've got our sample, we've got to start thinking about how to analyse the DNA. At the moment it's locked up inside the bacteria and we can't get at it. So we need to pop (lyse) the bacteria. This sounds simple, but not all bacteria have the same anatomy. Some of them have tough cell walls (generally Gram-positive bacteria), and others are only protected by delicate cell membranes (generally Gram-negative bacteria).

To lyse bacteria, we can either use mechanical, chemical or enzymatic techniques. The success of chemical and enzymatic methods can vary greatly depending on the sample. The pH and other chemical conditions can affect the performance of chemical and enzymatic methods, so they're not brilliant for environmental samples (8)⁠. The most commonly used mechanical technique is beat beating, which does exactly what it says. The sample is vigorously shaken in the presence of small beads. The problem with this method is that the beads don't differentiate between cell walls, cell membranes and DNA. So once DNA has been released from the safety of its bacteria, it can be beaten up and broken down by the beads as well. This is called DNA shearing. The amount of DNA shearing depends on factors such as the time and intensity of beating; the size of the beads and the bead to sample ratio. It's important to keep DNA shearing to a minimum as small fragments of DNA can increase PCR artefacts (we'll come to that later!) (8)⁠. 

0.5mm Silica beads used in bead beating.
Photo by: Lilly_M, Zirconia-silica-bead, CC BY-SA 3.0

Luckily, there's a way round this. A repeated bead-beating (RBB) protocol has been developed which can help limit DNA shearing. First, you perform an initial round of bead beating. This lyses the more delicate Gram-negative bacteria. You can then remove the liquid portion (the lysate), which will contain the DNA from any lysed bacteria, before a second round of beat-beating takes places to lyse  the more resistant Gram-positive bacteria and archaea. This minimises the amount of damage done to DNA from fragile bacteria which would otherwise be subjected to extra beating (9)⁠. When compared to other methods, RBB has been shown to have a superior DNA extraction efficiency and provide the best bacterial diversity, especially with respect to archaea and Gram-positive bacteria (10,11)⁠.


After cell lysis, the DNA has to be extracted. A wide variety of commercial kits are available, and each can introduce its own bias in terms of bacterial diversity and quality of DNA extracted (11,12)⁠. This makes the comparison of studies examining the microbiome difficult, as many will have used different DNA extraction techniques. There's also lots of home brew recipes available, but no one has done any comparative research with these. Apajalahti et al. described a method for lysis and extraction of DNA from ileal and caecal samples with reported cell lysis rates of >95% and > 99% respectively (13)⁠.

The final step of DNA extraction is purification which aims to remove PCR-inhibiting substances such as Dnases, polysaccharides and proteases which can interfere with the amplification of DNA (14)⁠. PCR inhibitors are present in chicken faecal and caecal samples, with a higher level detected in caecal samples, and they've also been found in human faeces (15)⁠. Commercially available DNA extraction kits contain steps which will help remove PCR inhibitors from samples (16)⁠, however there are additional steps which can be taken. The addition of non-acetylated bovine serum albumin (BSA) has been shown to partially overcome this inhibition, while polyethylene glycol (PEG) also facilitates PCR of faecal samples (15)⁠. T4 gene 32 protein has also been reported to reduce the inhibitory effects of contaminants (17)⁠.

Once DNA extraction is complete, you can estimate the quality of extracted DNA using agarose gel electrophoresis (18)⁠ and quantity using a spectrophotometre such as Nanodrop⁠. This step is especially important as DNA concentrations of a few to tens of picograms can cause random changes in PCR efficiency (19)⁠.

Once you've extracted you're DNA you're ready for the next step in the process, amplifying the DNA using PCR.

1. Park SH, Lee SI, Ricke SC. Microbial populations in naked neck chicken ceca raised on pasture flock fed with commercial yeast cell wall prebiotics via an Illumina MiSeq platform. PLoS One. 2016;11(3):1–15. [PDF]
2. Ballou AL, Ali RA, Mendoza MA, Ellis JC, Hassan HM, Croom WJ, et al. Development of the Chick Microbiome: How Early Exposure Influences Future Microbial Diversity. Front Vet Sci 2016; 3:2.  [PDF]

3. Zhu XY, Zhong T, Pandya Y, Joerger RD. 16S rRNA-based analysis of microbiota from the cecum of broiler chickens. Appl Environ Microbiol. 2002;68(1):124–37. [PDF]
4. Amit-Romach E, Sklan D, Uni Z. Microflora Ecology of the Chicken Intestine Using 16S Ribosomal DNA Primers. Poult Sci. 2004;83:1093–8. [PDF]

5. Thomas V, Clark J, Doré J. Fecal microbiota analysis: an overview of sample collection methods and sequencing strategies. Future Microbiol. 2015;10(9):1485–504. [Abstract]

6. Fouhy F, Deane J, Rea MC, O’Sullivan Ó, Ross RP, O’Callaghan G, et al. The effects of freezing on faecal microbiota as determined using Miseq sequencing and culture-based investigations. PLoS One. 2015;10(3):1–13. [PDF]

7. Bahl MI, Bergström A, Licht TR. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis. FEMS Microbiol Lett. 2012;329(2):193–7. [PDF]

8. Lauber CL, Zhou N, Gordon JI, Knight R, Fierer N. Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiol Lett. 2010;307(1):80–6. [PDF]

9. Stackebrandt E, Pukall R, Ulrichs G, Rheims H. Analysis of 16S rDNA clone libraries: part of the big picture. Proc 8th Int Symp Microb Ecol Microb Biosyst new Front Atl Canada Soc Microb Ecol Halifax, Nov Scotia, Canada. [PDF]

10. Osborn M a, Smith CJ. Molecular Microbial Ecology. Vol. 51. 2009. 370 p.

11.  Yu Z, Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques. 2004;36(5):808–12. [PDF]

12. Salonen A, Nikkilä J, Jalanka-Tuovinen J, Immonen O, Rajilić-Stojanović M, Kekkonen RA, et al. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis. J Microbiol Methods. 2010;81(2):127–34. [PDF]

13. Claassen S, du Toit E, Kaba M, Moodley C, Zar HJ, Nicol MP. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. J Microbiol Methods. 2013;94(2):103–10. [PDF]

14. Mirsepasi H, Persson S, Struve C, Andersen LOB, Petersen AM, Krogfelt K. Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA extraction compared to QIAamp DNA stool mini kit extraction. BMC Res Notes. 2014; 7:50. [PDF]

15. Apajalahti JH, Särkilahti LK, Mäki BR, Heikkinen JP, Nurminen PH, Holben WE. Effective recovery of bacterial DNA and percent-guanine-plus-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens. Appl Environ Microbiol. 1998;64(10):4084–8. [PDF]

16. Wilson IG. Inhibition and Facilitation of Nucleic Acid Amplification. Appl Environ Microbiol. 1997;63(10):3741–51. [PDF]

17. Rudi K, Høidal HK, Katla T, Johansen BK, Nordal J, Jakobsen KS. Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification. Society. 2004;70(2):790–7. [PDF]

18. Grant W, Long P. Environmental microbiology; Methods and Protocols. 2014. 241 p.

19. Chandler DP, Fredrickson JK, Brockman FJ. Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Mol Ecol. 1997;6(5):475–82. [PDF]

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