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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

Alignment identity, length and number of read of two FFPE samples against each of the ten GenBank H. pylori reference genomes and the culture samples of the two patients, respectively.
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pone-0026442-g002: Alignment identity, length and number of read of two FFPE samples against each of the ten GenBank H. pylori reference genomes and the culture samples of the two patients, respectively.

Mentions: In total, 241,126 and 201,709 sequence reads were obtained from the two FFPE samples, respectively. Among them, 14,532 (6.0%) and 9,830 (4.9%) could be mapped to contigs assembled from DNA of cultured H. pylori from the same patients (Culture 1 and Culture 2; Figure 2). When aligning FFPE sequences with each of the ten GenBank reference genomes, the numbers of aligned reads were only ∼11,000 and ∼8,700, respectively. The reference strains HPAG1, G27, P12, B8 and PeCan4 contain plasmids. Alignment of FFPE 1 sequences with these five strains resulted in on average 1,395 (12.1%) higher number of reads than with the five strains that lack plasmids (Figure 2). Alignment of raw sequences from Culture 1 also resulted in a greater (7.7%) number of reads matching to plasmid containing reference strains. No such difference was observed for sample FFPE 2 or Culture 2.


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)

Alignment identity, length and number of read of two FFPE samples against each of the ten GenBank H. pylori reference genomes and the culture samples of the two patients, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026442-g002: Alignment identity, length and number of read of two FFPE samples against each of the ten GenBank H. pylori reference genomes and the culture samples of the two patients, respectively.
Mentions: In total, 241,126 and 201,709 sequence reads were obtained from the two FFPE samples, respectively. Among them, 14,532 (6.0%) and 9,830 (4.9%) could be mapped to contigs assembled from DNA of cultured H. pylori from the same patients (Culture 1 and Culture 2; Figure 2). When aligning FFPE sequences with each of the ten GenBank reference genomes, the numbers of aligned reads were only ∼11,000 and ∼8,700, respectively. The reference strains HPAG1, G27, P12, B8 and PeCan4 contain plasmids. Alignment of FFPE 1 sequences with these five strains resulted in on average 1,395 (12.1%) higher number of reads than with the five strains that lack plasmids (Figure 2). Alignment of raw sequences from Culture 1 also resulted in a greater (7.7%) number of reads matching to plasmid containing reference strains. No such difference was observed for sample FFPE 2 or Culture 2.

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