Kristine M. Wylie

A physical map of the mouse genome

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human–mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.

A physical map of the human Y chromosome

The non-recombining region of the human Y chromosome (NRY), which comprises 95% of the chromosome, does not undergo sexual recombination and is present only in males. An understanding of its biological functions has begun to emerge from DNA studies of individuals with partial Y chromosomes, coupled with molecular characterization of genes implicated in gonadal sex reversal, Turner syndrome, graft rejection and spermatogenic failure. But mapping strategies applied successfully elsewhere in the genome have faltered in the NRY, where there is no meiotic recombination map and intrachromosomal repetitive sequences are abundant. Here we report a high-resolution physical map of the euchromatic, centromeric and heterochromatic regions of the NRY and its construction by unusual methods, including genomic clone subtraction and dissection of sequence family variants. Of the maps 758 DNA markers, 136 have multiple locations in the NRY, reflecting its unusually repetitive sequence composition. The markers anchor 1,038 bacterial artificial chromosome clones, 199 of which form a tiling path for sequencing.

A physical map of the chicken genome

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones. In an accompanying paper we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes.

Biogeography of the ecosystems of the healthy human body

BackgroundCharacterizing the biogeography of the microbiome of healthy humans is essential for understanding microbial associated diseases. Previous studies mainly focused on a single body habitat from a limited set of subjects. Here, we analyzed one of the largest microbiome datasets to date and generated a biogeographical map that annotates the biodiversity, spatial relationships, and temporal stability of 22 habitats from 279 healthy humans.ResultsWe identified 929 genera from more than 24 million 16S rRNA gene sequences of 22 habitats, and we provide a baseline of inter-subject variation for healthy adults. The oral habitat has the most stable microbiota with the highest alpha diversity, while the skin and vaginal microbiota are less stable and show lower alpha diversity. The level of biodiversity in one habitat is independent of the biodiversity of other habitats in the same individual. The abundances of a given genus at a body site in which it dominates do not correlate with the abundances at body sites where it is not dominant. Additionally, we observed the human microbiota exhibit both cosmopolitan and endemic features. Finally, comparing datasets of different projects revealed a project-based clustering pattern, emphasizing the significance of standardization of metagenomic studies.ConclusionsThe data presented here extend the definition of the human microbiome by providing a more complete and accurate picture of human microbiome biogeography, addressing questions best answered by a large dataset of subjects and body sites that are deeply sampled by sequencing.

Sequence Analysis of the Human Virome in Febrile and Afebrile Children

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF. We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.

Emerging view of the human virome

The human virome is the collection of all viruses that are found in or on humans, including both eukaryotic and prokaryotic viruses. Eukaryotic viruses clearly have important effects on human health, ranging from mild, self-limited acute or chronic infections to those with serious or fatal consequences. Prokaryotic viruses can also influence human health by affecting bacterial community structure and function. Therefore, definition of the virome is an important step toward understanding how microbes affect human health and disease. We review progress in virome analysis, which has been driven by advances in high-throughput, deep sequencing technology. Highlights from these studies include the association of viruses with clinical phenotypes and description of novel viruses that may be important pathogens. Together these studies indicate that analysis of the human virome is critical as we aim to understand how microbial communities influence human health and disease. Descriptions of the human virome will stimulate future work to understand how the virome affects long-term human health, immunity, and response to coinfections. Analysis of the virome ultimately may affect the treatment of patients with a variety of clinical syndromes.

Metagenomic analysis of double-stranded DNA viruses in healthy adults

BackgroundThe Human Microbiome Project (HMP) was undertaken with the goal of defining microbial communities in and on the bodies of healthy individuals using high-throughput metagenomic sequencing analysis. The viruses present in these microbial communities, the `human virome,’ are an important aspect of the human microbiome that is particularly understudied in the absence of overt disease. We analyzed eukaryotic double-stranded DNA (dsDNA) viruses, together with dsDNA replicative intermediates of single-stranded DNA viruses, in metagenomic sequence data generated by the HMP. We studied 706 samples from 102 subjects were studied, with each subject sampled at up to five major body habitats: nose, skin, mouth, vagina, and stool. Fifty-one individuals had samples taken at two or three time points 30 to 359 days apart from at least one of the body habitats.ResultsWe detected an average of 5.5 viral genera in each individual. At least one virus was detected in 92% of the individuals sampled. These viruses included herpesviruses, papillomaviruses, polyomaviruses, adenoviruses, anelloviruses, parvoviruses, and circoviruses. Each individual had a distinct viral profile, demonstrating the high interpersonal diversity of the virome. Some components of the virome were stable over time.ConclusionsThis study is the first to use high-throughput DNA sequencing to describe the diversity of eukaryotic dsDNA viruses in a large cohort of normal individuals who were sampled at multiple body sites. Our results show that the human virome is a complex component of the microbial flora. Some viruses establish long-term infections that may be associated with increased risk or possibly with protection from disease. A better understanding of the composition and dynamics of the virome may hold important keys to human health.

The "most wanted" taxa from the human microbiome for whole genome sequencing.

The goal of the Human Microbiome Project (HMP) is to generate a comprehensive catalog of human-associated microorganisms including reference genomes representing the most common species. Toward this goal, the HMP has characterized the microbial communities at 18 body habitats in a cohort of over 200 healthy volunteers using 16S rRNA gene (16S) sequencing and has generated nearly 1,000 reference genomes from human-associated microorganisms. To determine how well current reference genome collections capture the diversity observed among the healthy microbiome and to guide isolation and future sequencing of microbiome members, we compared the HMP’s 16S data sets to several reference 16S collections to create a ‘most wanted’ list of taxa for sequencing. Our analysis revealed that the diversity of commonly occurring taxa within the HMP cohort microbiome is relatively modest, few novel taxa are represented by these OTUs and many common taxa among HMP volunteers recur across different populations of healthy humans. Taken together, these results suggest that it should be possible to perform whole-genome sequencing on a large fraction of the human microbiome, including the ‘most wanted’, and that these sequences should serve to support microbiome studies across multiple cohorts. Also, in stark contrast to other taxa, the ‘most wanted’ organisms are poorly represented among culture collections suggesting that novel culture- and single-cell-based methods will be required to isolate these organisms for sequencing.

Novel Bacterial Taxa in the Human Microbiome

The human gut harbors thousands of bacterial taxa. A profusion of metagenomic sequence data has been generated from human stool samples in the last few years, raising the question of whether more taxa remain to be identified. We assessed metagenomic data generated by the Human Microbiome Project Consortium to determine if novel taxa remain to be discovered in stool samples from healthy individuals. To do this, we established a rigorous bioinformatics pipeline that uses sequence data from multiple platforms (Illumina GAIIX and Roche 454 FLX Titanium) and approaches (whole-genome shotgun and 16S rDNA amplicons) to validate novel taxa. We applied this approach to stool samples from 11 healthy subjects collected as part of the Human Microbiome Project. We discovered several low-abundance, novel bacterial taxa, which span three major phyla in the bacterial tree of life. We determined that these taxa are present in a larger set of Human Microbiome Project subjects and are found in two sampling sites (Houston and St. Louis). We show that the number of false-positive novel sequences (primarily chimeric sequences) would have been two orders of magnitude higher than the true number of novel taxa without validation using multiple datasets, highlighting the importance of establishing rigorous standards for the identification of novel taxa in metagenomic data. The majority of novel sequences are related to the recently discovered genus Barnesiella, further encouraging efforts to characterize the members of this genus and to study their roles in the microbial communities of the gut. A better understanding of the effects of less-abundant bacteria is important as we seek to understand the complex gut microbiome in healthy individuals and link changes in the microbiome to disease.

Enhanced virome sequencing using targeted sequence capture.

Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viral populations. It is culture independent, requires no a priori knowledge of the viruses in the sample, and may provide useful genomic information. However, MSS can lack sensitivity and may yield insufficient data for detailed analysis. We have created a targeted sequence capture panel, ViroCap, designed to enrich nucleic acid from DNA and RNA viruses from 34 families that infect vertebrate hosts. A computational approach condensed ∼1 billion bp of viral reference sequence into <200 million bp of unique, representative sequence suitable for targeted sequence capture. We compared the effectiveness of detecting viruses in standard MSS versus MSS following targeted sequence capture. First, we analyzed two sets of samples, one derived from samples submitted to a diagnostic virology laboratory and one derived from samples collected in a study of fever in children. We detected 14 and 18 viruses in the two sets, comprising 19 genera from 10 families, with dramatic enhancement of genome representation following capture enrichment. The median fold-increases in percentage viral reads post-capture were 674 and 296. Median breadth of coverage increased from 2.1% to 83.2% post-capture in the first set and from 2.0% to 75.6% in the second set. Next, we analyzed samples containing a set of diverse anellovirus sequences and demonstrated that ViroCap could be used to detect viral sequences with up to 58% variation from the references used to select capture probes. ViroCap substantially enhances MSS for a comprehensive set of viruses and has utility for research and clinical applications.