| 1. |
- Herlemann, Daniel P. R., et al.
(författare)
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Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria
- 2013
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Ingår i: mBio. - 2161-2129 .- 2150-7511. ; 4:3, s. e00569-12-
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Tidskriftsartikel (refereegranskat)abstract
- The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype's distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates. IMPORTANCE The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem.
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| 2. |
- Huang, Jiaqi, et al.
(författare)
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Rapid Screening of Complex DNA Samples by Single-Molecule Amplification and Sequencing
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:5, s. e19723-
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Tidskriftsartikel (refereegranskat)abstract
- Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample.
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| 3. |
- Huang, Jiaqi, et al.
(författare)
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Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma.
- 2015
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 61:11, s. 1408-16
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Variant profiling is crucial for developing personalized treatment and elucidating the etiology of this disease.METHODS: Patients with PDAC undergoing surgery from 2007 to 2012 (n = 73) were followed from diagnosis until death or the end of the study. We applied an anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) method to a panel of 65 selected genes and assessed analytical performance by sequencing a quantitative multiplex DNA reference standard. In clinical PDAC samples, detection of low-level KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations was validated by allele-specific PCR and digital PCR. We compared overall survival of patients according to KRAS mutation status by log-rank test and applied logistic regression to evaluate the association between smoking and tumor variant types.RESULTS: The AMP-based NGS method could detect variants with allele frequencies as low as 1% given sufficient sequencing depth (>1500×). Low-frequency KRAS G12 mutations (allele frequency 1%-5%) were all confirmed by allele-specific PCR and digital PCR. The most prevalent genetic alterations were in KRAS (78% of patients), TP53 (tumor protein p53) (25%), and SMAD4 (SMAD family member 4) (8%). Overall survival in T3-stage PDAC patients differed among KRAS mutation subtypes (P = 0.019). Transversion variants were more common in ever-smokers than in never-smokers (odds ratio 5.7; 95% CI 1.2-27.8).CONCLUSIONS: The AMP-based NGS method is applicable for profiling tumor variants. Using this approach, we demonstrated that in PDAC patients, KRAS mutant subtype G12V is associated with poorer survival, and that transversion variants are more common among smokers.
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| 4. |
- Willing, Ben P., et al.
(författare)
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A Pyrosequencing Study in Twins Shows That Gastrointestinal Microbial Profiles Vary With Inflammatory Bowel Disease Phenotypes
- 2010
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Ingår i: Gastroenterology. - : Elsevier BV. - 0016-5085 .- 1528-0012. ; 139:6, s. 1844-U105
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND & AIMS: The composition of the gastrointestinal microbiota is thought to have an important role in the etiology of inflammatory bowel diseases (IBDs) such as Crohn's disease (CD) and ulcerative colitis (UC). Interindividual variation and an inability to detect less abundant bacteria have made it difficult to correlate specific bacteria with disease. METHODS: We used 454 pyrotag sequencing to determine the compositions of microbial communities in feces samples collected from a cohort of 40 twin pairs who were concordant or discordant for CD or UC, and in mucosal samples from a subset of the cohort. The cohort primarily comprised patients who were in remission, but also some with active disease. RESULTS: The profiles of the microbial community differed with disease phenotypes; relative amounts of bacterial populations correlated with IBD phenotypes. The microbial compositions of individuals with CD differed from those of healthy individuals, but were similar between healthy individuals and individuals with UC. Profiles from individuals with CD that predominantly involved the ileum differed from those with CD that predominantly involved the colon; several bacterial populations increased or decreased with disease type. Changes specific to patients with ileal CD included the disappearance of core bacteria, such as Faecalibacterium and Roseburia, and increased amounts of Enterobacteriaceae and Ruminococcus gnavus. CONCLUSIONS: Bacterial populations differ in abundance among individuals with different phenotypes of CD. Specific species of bacteria are associated with ileal CD; further studies should investigate their role in pathogenesis.
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| 5. |
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| 6. |
- Zheng, Zongli, et al.
(författare)
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A Method for Metagenomics of Helicobacter pylori from Archived Formalin-Fixed Gastric Biopsies Permitting Longitudinal Studies of Carcinogenic Risk
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:10, s. e26442-
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Tidskriftsartikel (refereegranskat)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 similar to 96.4% identity with culture DNA from the same patients, while only similar to 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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| 7. |
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| 8. |
- Zheng, Zongli
(författare)
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Molecular epidemiologic studies on helicobacter pylori infection and stomach cancer risk
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Helicobacter pylori (H. pylori) infection increases stomach cancer risk. The aim of this thesis was to study genetic susceptibility from the host and to develop molecular methods for future characterization of bacterial virulence factors in longitudinal cohorts. In Study I, we investigated the association between genetic variation in an O-glycan transferase encoding gene (a4GnT) and H. pylori infection and gastric cancer risk in a Polish population-based case–control study (273 gastric cancer patients and 377 controls). A haplotype at rs2622694–rs397266 was associated with H. pylori infection, with the A-A haplotype associated with a higher risk compared with the most frequent G-G haplotype (odds ratio 2.30; 95% confidence intervals 1.35– 3.92). Neither this haplotype nor the tagSNPs were associated with overall gastric cancer risk. In Study II, we characterized genomic evolution of H. pylori over 20 years in the stomach. Whole genome of 21 sequential isolates 20 years apart, from 7 patients, were sequenced using 454 sequencing platform. There were on average 260 point mutations (range 70 to 488) per isolate over 20 years, and 45 recombinations (range 18 to 92). Genes in the cell motility category were overrepresented in point mutations and recombinations. Specifically, mutations often affected genes involved in chemotaxis, vacuolating cytotoxin-like protein, restriction and type IV secretory pathway; and recombinations affected glycosyltransferase involved in lipopolysaccharide biosynthesis. The major form of single nucleotide substitutions was transition (85%) and the minor form was transversion (15%). Mutation was sequence context-dependent. Clinical samples are often precious and of trace amounts. In Study III, we developed novel methods for DNA shotgun library construction and quantification. As compared with the standard procedure, our double-stranded and Y library construction methods are simpler and more efficient. A highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) was developed to quantify the amount of effective library. We also demonstrated that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labor intensive and costly titration can be eliminated and trace amounts of starting material is applicable. Archived formalin-fixed and paraffin-embedded (FFPE) biopsies, coupled with long term follow-up, are valuable resources for molecular epidemiologic studies. Study IV presented a method based on laser capture micro-dissection and modified whole genome sequencing methods to obtain metagenomic profiles of H. pylori from 15-year old FFPE biopsy sections.
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| 9. |
- Zheng, Zongli, et al.
(författare)
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Titration-free 454 sequencing using Y adapters
- 2011
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 6:9, s. 1367-1376
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Tidskriftsartikel (refereegranskat)abstract
- We describe a protocol for construction and quantification of libraries for emulsion PCR (emPCR)-based sequencing platforms such as Roche 454 or Ion Torrent PGM. The protocol involves library construction using customized Y adapters, quantification using TaqMan-MGB (minor groove binder) probe-based quantitative PCR (qPCR) and calculation of an optimal template-to-bead ratio based on Poisson statistics, thereby avoiding the need for a laborious titration assay. Unlike other qPCR methods, the TaqMan-MGB probe specifically quantifies effective libraries in molar concentration and does not require specialized equipment. A single quality control step prior to emulsion PCR ensures that libraries contain no adapter dimers and have an optimal length distribution. The presented protocol takes similar to 7 h to prepare eight barcoded libraries from genomic DNA into libraries that are ready to use for full-scale emPCR. It will be useful, for example, to allow analyses of precious clinical samples and amplification-free metatranscriptomics.
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