Preservation of bacterial DNA in 10-year-old guaiac FOBT cards and FIT tubes

Albuquerque, Matheus Couto Furtado; Herwaarden, Yasmijn van; Kortman, Guus A. M.; Dutilh, Bas E.; Bisseling, Tanya M.; Boleij, Annemarie

doi:10.1136/jclinpath-2017-204592

Cited by 4 publications

(11 citation statements)

References 8 publications

(10 reference statements)

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“…Results on the short-term storage of samples with alternative methods do not necessarily translate into unbiased microbiota results in long-term storage, which is often the case in cohort biobanks. In two small pilots, microbiome profile remained consistent as late as 10 years following storage on FOBT card [26, 27] suggesting the suitability of FOBT card (or similar products) for long-term cohort biobanking.…”

Section: Discussionmentioning

confidence: 99%

Assessment of the impact of different fecal storage protocols on the microbiota diversity and composition: a pilot study

et al. 2019

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Background Fecal samples are currently the most commonly studied proxy for gut microbiota. The gold standard of sample handling and storage for microbiota analysis is maintaining the cold chain during sample transfer and immediate storage at − 80 °C. Gut microbiota studies in large-scale, population-based cohorts require a feasible sample collection protocol. We compared the effect of three different storage methods and mock shipment: immediate freezing at − 80 °C, in 95% ethanol stored at room temperature (RT) for 48 h, and on blood collection card stored at RT for 48 h, on the measured composition of fecal microbiota of eight healthy, female volunteers by sequencing the V4 region of the 16S rRNA gene on an Illumina MiSeq. Results Shared operational taxonomic units (OTUs) between different methods were 68 and 3% for OTUs > 0.01 and < 0.01% mean relative abundance within each group, respectively. α and β-diversity measures were not significantly impacted by different storage methods. With the exception of Actinobacteria, fecal microbiota profiles at the phylum level were not significantly affected by the storage method. Actinobacteria was significantly higher in samples collected on card compared to immediate freezing (1.6 ± 1.1% vs. 0.4 ± 0.2%, p = 0.005) mainly driven by expansion of Actinobacteria relative abundance in fecal samples stored on card in two individuals. There was no statistically significant difference at lower taxonomic levels tested. Conclusion Consistent results of the microbiota composition and structure for different storage methods were observed. Fecal collection on card could be a suitable alternative to immediate freezing for fecal microbiota analysis using 16S rRNA gene amplicon sequencing. Electronic supplementary material The online version of this article (10.1186/s12866-019-1519-2) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Assessment of the impact of different fecal storage protocols on the microbiota diversity and composition: a pilot study

et al. 2019

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“…The bacterial DNA isolation strategy involved bacterial DNA enrichment through human cell lysis and DNAse treatment (Figure 1, upper part), which was followed up by our previously optimized bead‐beating protocol (Figure 1, lower part) (Couto Furtado Albuquerque et al, 2017). Whereas the bead‐beating protocol remained unchanged throughout this paper, two alternative strategies were tested for bacterial DNA enrichment.…”

Section: Methodsmentioning

confidence: 99%

Optimized bacterial DNA isolation method for microbiome analysis of human tissues

et al. 2021

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Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, the presence of host DNA in tissue isolates has hampered the analysis of host‐associated bacteria. Here, we present a DNA isolation protocol for tissue, optimized on biopsies from resected human colons (~2–5 mm in size), which includes reduction of human DNA without distortion of relative bacterial abundance at the phylum level. We evaluated which concentrations of Triton and saponin lyse human cells and leave bacterial cells intact, in combination with DNAse treatment to deplete released human DNA. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5‐fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ‐Proteobacteria, and Actinobacteria assessed by qPCR. Our optimized protocol was validated in the setting of two large clinical studies on 521 in vivo acquired colon biopsies of 226 patients using shotgun metagenomics. The resulting bacterial profiles exhibited alpha and beta diversities that are similar to the diversities found by 16S rRNA amplicon sequencing. A direct comparison between shotgun metagenomics and 16S rRNA amplicon sequencing of 15 forceps tissue biopsies showed similar bacterial profiles and a similar Shannon diversity index between the sequencing methods. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of all bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

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“…While several strategies, protocols and commercial kits have been developed to tackle this problem, so far none of these considered all elements that we considered important for analysis of tissue microbiomes. In this study we developed a protocol, inspired by Molzym(33), Hasan et al (8), and the Human microbiome project (HMP) (21), that enriched bacterial DNA through selective lysis of host DNA with 0.0125% saponin and subsequent DNAse treatment. This resulted in a bacterial DNA isolate in which all bacterial subsets were represented, without inducing lysis of bacterial cells or skewing bacterial composition in clinical samples.…”

Section: Discussionmentioning

confidence: 99%

“…The bacterial DNA isolation strategy involved bacterial DNA enrichment through human cell lysis and DNAse treatment (see figure 1, upper part), which was followed up by our previously optimized beadbeating protocol (see figure 1, lower part) (21). Whereas the bead-beating protocol remained unchanged throughout this paper, two alternative strategies were tested for the bacterial DNA enrichment.…”

Section: Bacterial Dna Isolation Protocolmentioning

confidence: 99%

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Optimized DNA isolation method for microbiome analysis of human tissues

Bruggeling

Garza

Achouiti

et al. 2020

Preprint

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Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, developments in the field of tissue microbiomes have been hampered by the presence of host DNA in isolates which interferes with the analysis of the bacterial content. Here, we present a DNA isolation protocol from tissue samples including reduction of host DNA without distortion of microbial abundance profiles. We evaluated which concentrations of Triton and saponin lyse host cells and leave bacterial cells intact, which was combined with DNAse treatment to deplete released host DNA. We applied our protocol to extract microbial DNA from ex vivo and in vivo acquired human colon biopsies (∼2-5 mm in size) and assessed the relative abundance of bacterial and human DNA by qPCR. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ-Proteobacteria and Actinobacteria. Our protocol combined with shotgun metagenomic sequencing revealed a colon tissue microbiome profile with a Shannon diversity index of 3.2 and an UniFrac distance of 0.54, which is comparable to reported numbers based on amplicon sequencing. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of rigid Gram-positive bacteria without depleting the more sensitive Gram-negative bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

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Preservation of bacterial DNA in 10-year-old guaiac FOBT cards and FIT tubes

Cited by 4 publications

References 8 publications

Assessment of the impact of different fecal storage protocols on the microbiota diversity and composition: a pilot study

Assessment of the impact of different fecal storage protocols on the microbiota diversity and composition: a pilot study

Optimized bacterial DNA isolation method for microbiome analysis of human tissues

Optimized DNA isolation method for microbiome analysis of human tissues

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