An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects ( p = 2.5 3 10 -6), and we observed a positive association with lymph node metastasis.[Supplemental material is available for this article.] There are variations on the method, but the basic approach involves shotgun sequencing bulk DNA or RNA isolated from disease tissue, computational subtraction of all sequence reads recognized as human, and comparison of the residual reads to databases of known microbial sequences in order to identify microbial species present in the initial specimen. The method is complementary to traditional culture and histolology-based protocols, and new massively parallel sequencing technologies impart high sensitivity. At present the power of the method remains restricted by the content of microbial sequence databases, but with our increasing reach into microbial sequence space, the comprehensiveness of these data resources continues to improve. In oncology, the identification of a novel polyomavirus in Merkel Cell carcinoma (Feng et al. 2008) is a recent demonstration of the utility of a metagenomics approach.Colorectal carcinoma (CRC) is the fourth leading cause of cancer deaths, responsible for approximately 610,000 deaths per year worldwide (World Health Organization 2011). It is also one of the first and best genetically characterized cancers, and specific somatic mutations in oncogenes and tumor suppressor genes have been found that are associated with progression from adenomatous lesions (polyps) to invasive carcinoma (Vogelstein et al. 1988). The root cause of CRC is unclear, but inflammation is a well-recognized risk factor (Wu et al. 2009;McLean et al. 2011). Given the link between H. pylori-mediated inflammation and gastric cancer (Marshall and Warren 1984), we asked if inflammatory microorganisms are associated with other gastrointestinal (GI) cancers. We began to address this question by undertaking a metagenomic survey of colorectal carcinoma. ResultsTotal RNA was isolated from frozen sections of 11 matched pairs of colorectal carcinoma and adjacent normal tissue specimens. RNA was purified by host ribosomal sequence depletion, rather than poly(A) selection, in order to re...

Massively parallel sequencing is a useful approach for characterizing T-cell receptor diversity. However, immune receptors are extraordinarily difficult sequencing targets because any given receptor variant may be present in very low abundance and may differ legitimately by only a single nucleotide. We show that the sensitivity of sequence-based repertoire profiling is limited by both sequencing depth and sequencing accuracy. At two timepoints, 1 wk apart, we isolated bulk PBMC plus naïve (CD45RA+/CD45RO-) and memory (CD45RA-/CD45RO+) T-cell subsets from a healthy donor. From T-cell receptor beta chain (TCRB) mRNA we constructed and sequenced multiple libraries to obtain a total of 1.7 billion paired sequence reads. The sequencing error rate was determined empirically and used to inform a high stringency data filtering procedure. The error filtered data yielded 1,061,522 distinct TCRB nucleotide sequences from this subject which establishes a new, directly measured, lower limit on individual T-cell repertoire size and provides a useful reference set of sequences for repertoire analysis. TCRB nucleotide sequences obtained from two additional donors were compared to those from the first donor and revealed limited sharing (up to 1.1%) of nucleotide sequences among donors, but substantially higher sharing (up to 14.2%) of inferred amino acid sequences. For each donor, shared amino acid sequences were encoded by a much larger diversity of nucleotide sequences than were unshared amino acid sequences. We also observed a highly statistically significant association between numbers of shared sequences and shared HLA class I alleles.

T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 59-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3 b diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR b clonotypes and provides precise measurements of CDR3 b length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3 b nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5′RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB.

SummaryWeibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures (“quanta”) of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities.