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A method for metagenomics of Helicobacter pylori from archived formalin-fixed gastric biopsies permitting longitudinal studies of carcinogenic risk.

Zheng Z, Andersson AF, Ye W, Nyrén O, Normark S, Engstrand L - PLoS ONE (2011)

Bottom Line: About 82% and 60% of the predicted genes in the two genomes were captured by at least a single sequencing read.Along with sequences displaying high similarity to known H. pylori genes, novel and highly variant H. pylori sequences were identified in the FFPE sections by our physical enrichment approach, which would likely not have been detected by a sequence capture approach.The study demonstrates the feasibility of longitudinal metagenomic studies of H. pylori using decade-preserved FFPE biopsies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The human microbiota has come into focus in the search for component causes of chronic diseases, such as gastrointestinal cancers. Presumably long induction periods and altered local environments after disease onset call for the development of methods for characterization of microorganisms colonizing the host decades before disease onset. Sequencing of microbial genomes in old formalin-fixed and paraffin-embedded (FFPE) gastrointestinal biopsies provides a means for such studies but is still challenging. Here we report a method based on laser capture micro-dissection and modified Roche 454 high-throughput pyrosequencing to obtain metagenomic profiles of Helicobacter pylori. We applied this method to two 15 year old FFPE biopsies from two patients. Frozen homogenized biopsies from the same gastroscopy sessions were also available for comparison after re-culture of H. pylori. For both patients, H. pylori DNA dissected from FFPE sections had ~96.4% identity with culture DNA from the same patients, while only ~92.5% identity with GenBank reference genomes, and with culture DNA from the other patient. About 82% and 60% of the predicted genes in the two genomes were captured by at least a single sequencing read. Along with sequences displaying high similarity to known H. pylori genes, novel and highly variant H. pylori sequences were identified in the FFPE sections by our physical enrichment approach, which would likely not have been detected by a sequence capture approach. The study demonstrates the feasibility of longitudinal metagenomic studies of H. pylori using decade-preserved FFPE biopsies.

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Laser capture microdissection a) Immunohistochemistry identified H. pylori colonizing area; b) Microdissection using neighboring sections; c) Dissected samples and remains.
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pone-0026442-g001: Laser capture microdissection a) Immunohistochemistry identified H. pylori colonizing area; b) Microdissection using neighboring sections; c) Dissected samples and remains.

Mentions: From each of the FFPE biopsy blocks, six sections, each 5 µm thick, were cut with disposable and autoclaved microtome blade. The surface section was discarded to reduce contamination. The other 5 sections were placed on autoclaved de-ionized warm (about 45°C) water. To guide the microdissection of H. pylori on the adjacent de-paraffinated sections (Figure 1), the second and sixth sections were collected on glass slides for IHC staining of H. pylori. The IHC staining included a de-paraffination step by 3 rinses in xylene solution for 10 minutes each and re-hydration through sequential rinses in ethanol (99% 5 minutes twice; 95% for 2 minutes; 70% for 2 minutes) and in water for 2 minutes. Antigen retrieval was achieved through heat treatment at 95°C for 20 minutes in sodium-citrate buffer (pH 6.0), followed by non-specific background blocking in a solution containing 2% swine serum, 1% BSA and 0.1% azide in TBS pH 7.6 for 30 minutes. Primary antibody (polyclonal rabbit anti-H. pylori, DAKO, Denmark) 1∶1000 in blocking buffer was applied at ambient temperature (22°C) for 1 hour, then rinsed 3 times in fresh 1 x TBS, 0.5% Tween 20, each for 5 minutes. Endogenous peroxidase was blocked in 3% H2O2 for 10 minutes and rinsed in fresh 1 x TBS for 5 minutes. Secondary antibody (Swine anti-rabbit, biotinylated, DAKO) 1∶500 in 1 x TBS was applied and incubated at ambient temperature for 1 hour, and rinsed 3 times in fresh 1 x TBS, 0.5% Tween 20, each for 5 minutes. The sections were then incubated in strepavidin-biotin-peroxidase complex (DAKO) according to the manufacturer's instruction. The peroxidase was then developed by the 3,3′-diaminobenzidine tetrahydrochloride (DAB, Zymed), resulting in brown stained H. pylori.


A method for metagenomics of Helicobacter pylori from archived formalin-fixed gastric biopsies permitting longitudinal studies of carcinogenic risk.

Zheng Z, Andersson AF, Ye W, Nyrén O, Normark S, Engstrand L - PLoS ONE (2011)

Laser capture microdissection a) Immunohistochemistry identified H. pylori colonizing area; b) Microdissection using neighboring sections; c) Dissected samples and remains.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198776&req=5

pone-0026442-g001: Laser capture microdissection a) Immunohistochemistry identified H. pylori colonizing area; b) Microdissection using neighboring sections; c) Dissected samples and remains.
Mentions: From each of the FFPE biopsy blocks, six sections, each 5 µm thick, were cut with disposable and autoclaved microtome blade. The surface section was discarded to reduce contamination. The other 5 sections were placed on autoclaved de-ionized warm (about 45°C) water. To guide the microdissection of H. pylori on the adjacent de-paraffinated sections (Figure 1), the second and sixth sections were collected on glass slides for IHC staining of H. pylori. The IHC staining included a de-paraffination step by 3 rinses in xylene solution for 10 minutes each and re-hydration through sequential rinses in ethanol (99% 5 minutes twice; 95% for 2 minutes; 70% for 2 minutes) and in water for 2 minutes. Antigen retrieval was achieved through heat treatment at 95°C for 20 minutes in sodium-citrate buffer (pH 6.0), followed by non-specific background blocking in a solution containing 2% swine serum, 1% BSA and 0.1% azide in TBS pH 7.6 for 30 minutes. Primary antibody (polyclonal rabbit anti-H. pylori, DAKO, Denmark) 1∶1000 in blocking buffer was applied at ambient temperature (22°C) for 1 hour, then rinsed 3 times in fresh 1 x TBS, 0.5% Tween 20, each for 5 minutes. Endogenous peroxidase was blocked in 3% H2O2 for 10 minutes and rinsed in fresh 1 x TBS for 5 minutes. Secondary antibody (Swine anti-rabbit, biotinylated, DAKO) 1∶500 in 1 x TBS was applied and incubated at ambient temperature for 1 hour, and rinsed 3 times in fresh 1 x TBS, 0.5% Tween 20, each for 5 minutes. The sections were then incubated in strepavidin-biotin-peroxidase complex (DAKO) according to the manufacturer's instruction. The peroxidase was then developed by the 3,3′-diaminobenzidine tetrahydrochloride (DAB, Zymed), resulting in brown stained H. pylori.

Bottom Line: About 82% and 60% of the predicted genes in the two genomes were captured by at least a single sequencing read.Along with sequences displaying high similarity to known H. pylori genes, novel and highly variant H. pylori sequences were identified in the FFPE sections by our physical enrichment approach, which would likely not have been detected by a sequence capture approach.The study demonstrates the feasibility of longitudinal metagenomic studies of H. pylori using decade-preserved FFPE biopsies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The human microbiota has come into focus in the search for component causes of chronic diseases, such as gastrointestinal cancers. Presumably long induction periods and altered local environments after disease onset call for the development of methods for characterization of microorganisms colonizing the host decades before disease onset. Sequencing of microbial genomes in old formalin-fixed and paraffin-embedded (FFPE) gastrointestinal biopsies provides a means for such studies but is still challenging. Here we report a method based on laser capture micro-dissection and modified Roche 454 high-throughput pyrosequencing to obtain metagenomic profiles of Helicobacter pylori. We applied this method to two 15 year old FFPE biopsies from two patients. Frozen homogenized biopsies from the same gastroscopy sessions were also available for comparison after re-culture of H. pylori. For both patients, H. pylori DNA dissected from FFPE sections had ~96.4% identity with culture DNA from the same patients, while only ~92.5% identity with GenBank reference genomes, and with culture DNA from the other patient. About 82% and 60% of the predicted genes in the two genomes were captured by at least a single sequencing read. Along with sequences displaying high similarity to known H. pylori genes, novel and highly variant H. pylori sequences were identified in the FFPE sections by our physical enrichment approach, which would likely not have been detected by a sequence capture approach. The study demonstrates the feasibility of longitudinal metagenomic studies of H. pylori using decade-preserved FFPE biopsies.

Show MeSH
Related in: MedlinePlus