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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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Related in: MedlinePlus

Venn diagram showing the numbers of mapped reads from two FFPE samples against the ten H. pylori references (tenRef) and the culture samples of the two patients.
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pone-0026442-g003: Venn diagram showing the numbers of mapped reads from two FFPE samples against the ten H. pylori references (tenRef) and the culture samples of the two patients.

Mentions: Venn diagrams (Figure 3) show that there were higher numbers of FFPE sequences aligned with the culture sequences from the same patient than with the ten reference genomes combined or with the culture sequences from the other patient. In addition, the number of FFPE 1 reads that mapped uniquely to Culture 1 was higher than the numbers of FFPE 1 reads that mapped uniquely to tenRef or with Culture 2 (both P values <10−16). The number of FFPE 2 reads that mapped uniquely to Culture 2 was higher than the number of FFPE 2 mapped uniquely to Culture 1, but less than the tenRef (both P values <10−4). Annotation of the FFPE sequences aligning only with culture sequences from the same patient revealed an abundance of genes that are components of plasmid (relaxase, transposase, replicase, etc.) and of restriction-modification systems (see supplementary files File S1 and File S2.).


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)

Venn diagram showing the numbers of mapped reads from two FFPE samples against the ten H. pylori references (tenRef) and the culture samples of the two patients.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026442-g003: Venn diagram showing the numbers of mapped reads from two FFPE samples against the ten H. pylori references (tenRef) and the culture samples of the two patients.
Mentions: Venn diagrams (Figure 3) show that there were higher numbers of FFPE sequences aligned with the culture sequences from the same patient than with the ten reference genomes combined or with the culture sequences from the other patient. In addition, the number of FFPE 1 reads that mapped uniquely to Culture 1 was higher than the numbers of FFPE 1 reads that mapped uniquely to tenRef or with Culture 2 (both P values <10−16). The number of FFPE 2 reads that mapped uniquely to Culture 2 was higher than the number of FFPE 2 mapped uniquely to Culture 1, but less than the tenRef (both P values <10−4). Annotation of the FFPE sequences aligning only with culture sequences from the same patient revealed an abundance of genes that are components of plasmid (relaxase, transposase, replicase, etc.) and of restriction-modification systems (see supplementary files File S1 and File S2.).

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