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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

Simulations of sequencing depth versus yield of 1000-bp bins for the two laser capture microdissected samples (FFPE 1 and FFPE 2).Each data point was obtained through 1000 simulations, with replacement, where each simulation contained a specified number (the X axis) of randomly sampled sequences, increased in steps of 100 up to the total number of sequences available. The number of unique bins for each simulation dataset was calculated, as was the average number derived from 1000 simulation datasets. These average numbers were plotted. The two data points labeled where the curves start to level off approximately, as examples of reasonable sequencing depth for cost-effective consideration.
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pone-0026442-g005: Simulations of sequencing depth versus yield of 1000-bp bins for the two laser capture microdissected samples (FFPE 1 and FFPE 2).Each data point was obtained through 1000 simulations, with replacement, where each simulation contained a specified number (the X axis) of randomly sampled sequences, increased in steps of 100 up to the total number of sequences available. The number of unique bins for each simulation dataset was calculated, as was the average number derived from 1000 simulation datasets. These average numbers were plotted. The two data points labeled where the curves start to level off approximately, as examples of reasonable sequencing depth for cost-effective consideration.

Mentions: Figure 5 shows the relation between sequencing depth and yield of unique 1000-bp bins, assessed for each FFPE sample through simulations. The yield of unique bins increased with increasing sequencing depth, but around 5,000 sequence reads it approached a plateau, similarly for both of the samples.


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)

Simulations of sequencing depth versus yield of 1000-bp bins for the two laser capture microdissected samples (FFPE 1 and FFPE 2).Each data point was obtained through 1000 simulations, with replacement, where each simulation contained a specified number (the X axis) of randomly sampled sequences, increased in steps of 100 up to the total number of sequences available. The number of unique bins for each simulation dataset was calculated, as was the average number derived from 1000 simulation datasets. These average numbers were plotted. The two data points labeled where the curves start to level off approximately, as examples of reasonable sequencing depth for cost-effective consideration.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026442-g005: Simulations of sequencing depth versus yield of 1000-bp bins for the two laser capture microdissected samples (FFPE 1 and FFPE 2).Each data point was obtained through 1000 simulations, with replacement, where each simulation contained a specified number (the X axis) of randomly sampled sequences, increased in steps of 100 up to the total number of sequences available. The number of unique bins for each simulation dataset was calculated, as was the average number derived from 1000 simulation datasets. These average numbers were plotted. The two data points labeled where the curves start to level off approximately, as examples of reasonable sequencing depth for cost-effective consideration.
Mentions: Figure 5 shows the relation between sequencing depth and yield of unique 1000-bp bins, assessed for each FFPE sample through simulations. The yield of unique bins increased with increasing sequencing depth, but around 5,000 sequence reads it approached a plateau, similarly for both of the samples.

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