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Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants.

Hill CJ, Brown JR, Lynch DB, Jeffery IB, Ryan CA, Ross RP, Stanton C, O'Toole PW - Microbiome (2016)

Bottom Line: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing.After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2.We would recommend extraction ideally within the first week of storage.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork, Cork, Ireland.

ABSTRACT

Background: Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults.

Results: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p < 0.001). When the microbiota profiles were analysed at the operational taxonomic unit (OTU) level, three infant datasets in the study did not cluster together, while only one elderly dataset did not. The lower microbiota diversity of the infant gut microbiota compared to the elderly gut microbiota (p < 0.001) means that any alteration in the infant datasets has a proportionally larger effect.

Conclusions: The commercial storage vials appear to be suitable for high diversity microbiota samples, but may be less appropriate for lower diversity samples. Differences between fresh and stored samples mean that where storage is unavoidable, a consistent storage regime should be used. We would recommend extraction ideally within the first week of storage.

No MeSH data available.


Elderly subjects have higher diversity than infant subjects and are less variable after storage. a Shannon diversity of infant and elderly samples. b Shannon diversity values after storage across infant and elderly subject samples combined. c Higher variabilty of infant subjects demonstrated by increase in the absolute Spearman distances between samples. (i–iii) elderly samples and (iii–vi) infant samples. (i) and (iv) fresh vs 1 week’s storage; (ii) and (v) fresh vs 2 weeks’ storage; (iii) and (vi) 1 week’s storage vs 2 weeks’ storage
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Fig3: Elderly subjects have higher diversity than infant subjects and are less variable after storage. a Shannon diversity of infant and elderly samples. b Shannon diversity values after storage across infant and elderly subject samples combined. c Higher variabilty of infant subjects demonstrated by increase in the absolute Spearman distances between samples. (i–iii) elderly samples and (iii–vi) infant samples. (i) and (iv) fresh vs 1 week’s storage; (ii) and (v) fresh vs 2 weeks’ storage; (iii) and (vi) 1 week’s storage vs 2 weeks’ storage

Mentions: The microbiota diversity of the infant samples, as measured by the Shannon index, was much lower than that of the elderly subjects (Fig. 3a, p < 0.001). Storage of the samples had no effect on this diversity after either 1 or 2 weeks (Fig. 3b). The apparent infant microbiota composition exhibited significantly higher variation than that of elderly subjects’ samples, reflected in the Spearman distance between storage time points (Fig. 3c). The difference between stored samples at week 1 and week 2 in the elderly subjects (Fig. 3c(iii)) was significantly lower than the difference between fresh and either 1 week (Fig. 3c(i)) or 2 weeks’ (Fig. 3c(ii)) storage. The difference was consistently higher under all storage conditions in the infant samples. This demonstrated that individual infant samples tended to display greater difference between the microbiota composition of freshly extracted samples and those stored before extraction. This suggested that the lower diversity of the infant gut microbiota resulted in a higher susceptibility to non-specific changes in the profile of the gut microbiota after storage. The individual infant and elderly microbiota composition data are presented in Additional file 2: Figures S1 and Additional file 3: Figure S2, respectively.Fig. 3


Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants.

Hill CJ, Brown JR, Lynch DB, Jeffery IB, Ryan CA, Ross RP, Stanton C, O'Toole PW - Microbiome (2016)

Elderly subjects have higher diversity than infant subjects and are less variable after storage. a Shannon diversity of infant and elderly samples. b Shannon diversity values after storage across infant and elderly subject samples combined. c Higher variabilty of infant subjects demonstrated by increase in the absolute Spearman distances between samples. (i–iii) elderly samples and (iii–vi) infant samples. (i) and (iv) fresh vs 1 week’s storage; (ii) and (v) fresh vs 2 weeks’ storage; (iii) and (vi) 1 week’s storage vs 2 weeks’ storage
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4862223&req=5

Fig3: Elderly subjects have higher diversity than infant subjects and are less variable after storage. a Shannon diversity of infant and elderly samples. b Shannon diversity values after storage across infant and elderly subject samples combined. c Higher variabilty of infant subjects demonstrated by increase in the absolute Spearman distances between samples. (i–iii) elderly samples and (iii–vi) infant samples. (i) and (iv) fresh vs 1 week’s storage; (ii) and (v) fresh vs 2 weeks’ storage; (iii) and (vi) 1 week’s storage vs 2 weeks’ storage
Mentions: The microbiota diversity of the infant samples, as measured by the Shannon index, was much lower than that of the elderly subjects (Fig. 3a, p < 0.001). Storage of the samples had no effect on this diversity after either 1 or 2 weeks (Fig. 3b). The apparent infant microbiota composition exhibited significantly higher variation than that of elderly subjects’ samples, reflected in the Spearman distance between storage time points (Fig. 3c). The difference between stored samples at week 1 and week 2 in the elderly subjects (Fig. 3c(iii)) was significantly lower than the difference between fresh and either 1 week (Fig. 3c(i)) or 2 weeks’ (Fig. 3c(ii)) storage. The difference was consistently higher under all storage conditions in the infant samples. This demonstrated that individual infant samples tended to display greater difference between the microbiota composition of freshly extracted samples and those stored before extraction. This suggested that the lower diversity of the infant gut microbiota resulted in a higher susceptibility to non-specific changes in the profile of the gut microbiota after storage. The individual infant and elderly microbiota composition data are presented in Additional file 2: Figures S1 and Additional file 3: Figure S2, respectively.Fig. 3

Bottom Line: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing.After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2.We would recommend extraction ideally within the first week of storage.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork, Cork, Ireland.

ABSTRACT

Background: Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults.

Results: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p < 0.001). When the microbiota profiles were analysed at the operational taxonomic unit (OTU) level, three infant datasets in the study did not cluster together, while only one elderly dataset did not. The lower microbiota diversity of the infant gut microbiota compared to the elderly gut microbiota (p < 0.001) means that any alteration in the infant datasets has a proportionally larger effect.

Conclusions: The commercial storage vials appear to be suitable for high diversity microbiota samples, but may be less appropriate for lower diversity samples. Differences between fresh and stored samples mean that where storage is unavoidable, a consistent storage regime should be used. We would recommend extraction ideally within the first week of storage.

No MeSH data available.