A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

Comparison of Major and Minor Viral SNPs Identified through Single Template Sequencing and Pyrosequencing in Acute HIV-1 Infection.

Massively parallel sequencing (MPS) technologies, such as 454-pyrosequencing, allow for the identification of variants in sequence populations at lower levels than consensus sequencing and most single-template Sanger sequencing experiments. We sought to determine if the greater depth of population sampling attainable using MPS technology would allow detection of minor variants in HIV founder virus populations very early in infection in instances where Sanger sequencing detects only a single variant. We compared single nucleotide polymorphisms (SNPs) during acute HIV-1 infection from 32 subjects using both single template Sanger and 454-pyrosequencing. Pyrosequences from a median of 2400 viral templates per subject and encompassing 40% of the HIV-1 genome, were compared to a median of five individually amplified near full-length viral genomes sequenced using Sanger technology. There was no difference in the consensus nucleotide sequences over the 3.6kb compared in 84% of the subjects infected with single founders and 33% of subjects infected with multiple founder variants: among the subjects with disagreements, mismatches were found in less than 1% of the sites evaluated (of a total of nearly 117,000 sites across all subjects). The majority of the SNPs observed only in pyrosequences were present at less than 2% of the subject's viral sequence population. These results demonstrate the utility of the Sanger approach for study of early HIV infection and provide guidance regarding the design, utility and limitations of population sequencing from variable template sources, and emphasize parameters for improving the interpretation of massively parallel sequencing data to address important questions regarding target sequence evolution.

Quality score based identification and correction of pyrosequencing errors.

Massively-parallel DNA sequencing using the 454/pyrosequencing platform allows in-depth probing of diverse sequence populations, such as within an HIV-1 infected individual. Analysis of this sequence data, however, remains challenging due to the shorter read lengths relative to that obtained by Sanger sequencing as well as errors introduced during DNA template amplification and during pyrosequencing. The ability to distinguish real variation from pyrosequencing errors with high sensitivity and specificity is crucial to interpreting sequence data. We introduce a new algorithm, CorQ (Correction through Quality), which utilizes the inherent base quality in a sequence-specific context to correct for homopolymer and non-homopolymer insertion and deletion (indel) errors. CorQ also takes uneven read mapping into account for correcting pyrosequencing miscall errors and it identifies and corrects carry forward errors. We tested the ability of CorQ to correctly call SNPs on a set of pyrosequences derived from ten viral genomes from an HIV-1 infected individual, as well as on six simulated pyrosequencing datasets generated using non-zero error rates to emulate errors introduced by PCR. When combined with the AmpliconNoise error correction method developed to remove ambiguities in signal intensities, we attained a 97% reduction in indel errors, a 98% reduction in carry forward errors, and >97% specificity of SNP detection. When compared to four other error correction methods, AmpliconNoise+CorQ performed at equal or higher SNP identification specificity, but the sensitivity of SNP detection was consistently higher (>98%) than other methods tested. This combined procedure will therefore permit examination of complex genetic populations with improved accuracy.

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

Unique signatures of long noncoding RNA expression in response to virus infection and altered innate immune signaling.

Studies of the host response to virus infection typically focus on protein-coding genes. However, non-protein-coding RNAs (ncRNAs) are transcribed in mammalian cells, and the roles of many of these ncRNAs remain enigmas. Using next-generation sequencing, we performed a whole-transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection across four founder mouse strains of the Collaborative Cross. We observed differential expression of approximately 500 annotated, long ncRNAs and 1,000 nonannotated genomic regions during infection. Moreover, studies of a subset of these ncRNAs and genomic regions showed the following. (i) Most were similarly regulated in response to influenza virus infection. (ii) They had distinctive kinetic expression profiles in type I interferon receptor and STAT1 knockout mice during SARS-CoV infection, including unique signatures of ncRNA expression associated with lethal infection. (iii) Over 40% were similarly regulated in vitro in response to both influenza virus infection and interferon treatment. These findings represent the first discovery of the widespread differential expression of long ncRNAs in response to virus infection and suggest that ncRNAs are involved in regulating the host response, including innate immunity. At the same time, virus infection models provide a unique platform for studying the biology and regulation of ncRNAs.

Digital genome-wide ncRNA expression, including SnoRNAs, across 11 human tissues using polyA-neutral amplification.

Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available.

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing.

BACKGROUND: DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. RESULTS: We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. CONCLUSION: The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.

Eukaryotic signature proteins of Prosthecobacter dejongeii and Gemmata sp. Wa-1 as revealed by in silico analysis.

The genomes of representatives of three bacterial phyla have been compared with the list of 347 eukaryotic signature proteins (ESPs) derived by Hartman and Fedorov [Proc. Natl. Acad. Sci. USA 99 (2002) 1420]. The species included Prosthecobacter dejongeii of the Verrucomicrobia phylum, Gemmata sp. Wa-1 of the Planctomycetes phylum and Caulobacter crescentus of the Proteobacteria. The protist Trypanosoma brucei was used as a eukaryotic control. P. dejongeii had unique ERGO blast matches to alpha-, beta-, and gamma-tubulin, to Set2, a transcriptional factor associated with eukaryotic DNA, and to LAMMER protein kinase for a total of 10 high-scoring ESP matches altogether. Gemmata sp. Wa-1 shared four of its 17 high-scoring ESP matches with P. dejongeii, and that information coupled with other genomic data provides strong support that these two phyla are related to one another. If the ESP list is an accurate listing of unique eukaryotic proteins, then the low number of high-scoring matches between the proteins of these two bacteria with the list raises doubts about these phyla being direct ancestors of the Eucarya. However, this does not rule out the possibility that ancestral members of either the Verrucomicrobia or Planctomycetes may have played an important role in the evolution of a proto-eukaryotic organism.