The Sensitivity of Massively Parallel Sequencing for Detecting Candidate Infectious Agents Associated with Human Tissue

Moore, Richard A.; Warren, René L.; Freeman, Jamie; Gustavsen, Julia; Chénard, Caroline; Friedman, Jan M.; Suttle, Curtis A.; Zhao, Yongjun; Holt, Robert A.

doi:10.1371/journal.pone.0019838

Cited by 61 publications

(58 citation statements)

References 16 publications

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“…Illumina RNA-seq libraries were constructed, barcoded, and pooled, and two lanes of paired-end sequencing data were obtained using the Illumina GAIIx platform. Reads were filtered for base quality and low complexity, then aligned pairwise to human rRNA and cDNA (Flicek et al 2011) and genome (hg18) reference sequences using Burrows-Wheeler Aligner (BWA) (Li and Durbin 2010), as previously described (Moore et al 2011). Aligned reads were removed from the data set, leaving 34.9 million pairs (Supplemental Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…Eleven colorectal tumor samples and 11 matched normal samples were processed, as detailed previously (Moore et al 2011), using an RNeasy Plus mini kit (QIAGEN) to purify total RNA or an AllPrep DNA/RNA mini kit (QIAGEN) to purify both DNA and RNA. RNA quality and concentration were assessed using Agilent Bioanalyzer 2000 RNA Nanochips.…”

Section: Metagenomic Library Construction and Sequencingmentioning

confidence: 99%

“…Those that have, such as Human Papilloma Virus, Hepatitis B and C virus, and Helicobacter pylori, alone are responsible for an estimated 15% of the global cancer burden, based on strength of the association and prevalence of infection (Parkin 2006). Metagenomics methods developed over the past decade (Weber et al 2002;Moore et al 2011) provide a useful approach to identifying microbial sequence signatures in diseases that have a possible or suspected infectious etiology. 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.…”

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Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma

Castellarin

Warren²,

Freeman³

et al. 2011

Genome Res.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Metagenomic Library Construction and Sequencingmentioning

confidence: 99%

mentioning

confidence: 99%

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Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma

Castellarin

Warren²,

Freeman³

et al. 2011

Genome Res.

Self Cite

1,651

1,344

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“…In contrast to metagenomic analysis using pyrosequencing, Illumina short-read sequencing enables a greater depth (by an order of magnitude) that is reflected in a very low detection limit. A recent report revealed that viral transcripts could be found at frequencies of Ͻ1 in 1,000,000 (28). However, because of short reads, de novo assembly without any reference is difficult to conduct, so it is not suitable for discovering an unknown viral genome.…”

Section: Discussionmentioning

confidence: 99%

Use of Illumina Deep Sequencing Technology To Differentiate Hepatitis C Virus Variants

et al. 2012

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Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus that shows diverse viral populations even in one individual. Though Sanger sequencing has been used to determine viral sequences, deep sequencing technologies are much faster and can perform large-scale sequencing. We demonstrate the successful use of Illumina deep sequencing technology and subsequent analyses to determine the genetic variants and amino acid substitutions in both treatment-naïve (patient 1) and treatmentexperienced (patient 7) isolates from HCV-infected patients. As a result, almost the full nucleotide sequence of HCV was detectable for patients 1 and 7. The reads were mapped to the HCV reference sequence. The coverage was 99.8% and the average depth was 69.5؋ for patient 7, with values of 99.4% (coverage) and 51.1؋ (average depth) for patient 1. In patient 7, amino acid (aa) 70 in the core region showed arginine, with methionine at aa 91, by Sanger sequencing. Major variants showed the same amino acid sequence, but minor variants were detectable in 18% ( . The single open reading frame encodes a polyprotein of 3,011 amino acids (aa) that is cleaved by viral and cellular proteases into 10 different proteins. The three structural proteins, which constitute the viral particle, include the core protein and the envelope glycoproteins E1 and E2. Two regions in E2, known as hypervariable regions 1 and 2, are reported to have extreme sequence variability. The seven nonstructural components include the p7 polypeptide, the NS2-3 protease, the NS3 serine protease and RNA helicase, the NS4A polypeptide, the NA4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (RdRp) (29). At both ends of the open reading frame lie the 5=-and 3=-untranslated regions (5=-UTR and 3=-UTR). The nucleotide sequence of the 5=-UTR is relatively well conserved among different HCV genotypes. The HCV 5=-UTR contains an internal ribosome entry site (IRES) that directs the cap-independent initiation of virus translation and forms on four characteristic stem-loop structures (17, 18). HCV displays very high genetic variability both in populations and within infected individuals, where it exists as a cluster of closely related but distinct variants, termed "quasispecies," as occurs in many other RNA viruses with a polymerase enzyme lacking proofreading ability (6,8,26).Current standard treatment of chronic HCV infection is based on the combination of pegylated alpha interferon (peg-IFN-␣) and ribavirin (RBV). However, patients with a high load of genotype 1b virus (Ͼ1 ϫ 10 5 log IU/ml) do not achieve high sustained virological response (SVR) rates (Ͻ50%), even when the most effective combination treatment (IFN plus RBV) is administered for 48 weeks (14,25). Some investigations concerning therapeutic prediction based on virological features revealed that substitutions of arginine for glutamine at amino acid (aa) 70 and/or leucine for methionine at aa 91 in the core region are independent and significant factors associated with SVR or that patients whose viruses ...

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“…Although Fusobacterium species are part of the gut microbiome in human, their invasive [1,2], adherent [3,4], and pro-inflammatory [5][6][7] features have been noted. Fusobacterium have been associated with inflammatory disorders such as periodontitis [8], cerebral abscesses [9], acute appendicitis [10] and inflammatory bowel diseases [1,11,12].…”

Section: Introductionmentioning

confidence: 99%