High-resolution Whole-genome Sequencing Research Articles

Using a new analytic approach for genotyping and phenotyping chromosome 9p deletion syndrome.

Using a new analytic method ("unique non-overlapping region" (UNOR) analysis), we characterized the genotypes and phenotypes of a large cohort of individuals diagnosed with chromosome 9p deletion syndrome (9PMS) and defined critical genomic regions. We extracted phenotypic information from 48 individuals with 9PMS from medical records and used a guided interview with caregivers to clarify ambiguities. Using high-resolution whole-genome sequencing for breakpoint definition, we aligned deletions and drew virtual breakpoints to obtain UNORs associated with phenotypic characteristics. We next extracted genotype and phenotype data for 57 individuals identified from a systematic review of the 9PMS literature and analyzed these as above. Common phenotypic features included developmental delay/intellectual disability, dysmorphic features, hypotonia, genital defects in XY individuals, psychiatric diagnoses, chronic constipation, atopic disease, vision problems, autism spectrum disorder, gastroesophageal reflux disease, trigonocephaly, congenital heart disease, and neonatal hypoglycemia. Our approach confirmed previous literature reports of an association of FREM1 with trigonocephaly and suggested a possible modifier element for this phenotype. In conclusion, the UNOR approach delineated phenotypic characteristics for 9PMS and confirmed the critical role of FREM1 and a possible long-distance regulatory element in pathogenesis of trigonocephaly that will need to be replicated in future studies.

Human microbiota drives hospital-associated antimicrobial resistance dissemination in the urban environment and mirrors patient case rates

BackgroundThe microbial community composition of urban environments is primarily determined by human activity. The use of metagenomics to explore how microbial communities are shaped in a city provides a novel input that can improve decisions on public health measures, architectural design, and urban resilience. Of note, the sewage system in a city acts as a complex reservoir of bacteria, pharmaceuticals, and antimicrobial resistant (AMR) genes that can be an important source of epidemiological information. Hospital effluents are rich in patient-derived bacteria and can thus readily become a birthplace and hotspot reservoir for antibiotic resistant pathogens which are eventually incorporated into the environment. Yet, the scope to which nosocomial outbreaks impact the urban environment is still poorly understood.ResultsIn this work, we extensively show that different urban waters from creeks, beaches, sewage spillways and collector pipes enclose discrete microbial communities that are characterized by a differential degree of contamination and admixture with human-derived bacteria. The abundance of human bacteria correlates with the abundance of AMR genes in the environment, with beta-lactamases being the top-contributing class to distinguish low vs. highly-impacted urban environments. Indeed, the abundance of beta-lactamase resistance and carbapenem resistance determinants in the urban environment significantly increased in a 1-year period. This was in line with a pronounced increase of nosocomial carbapenem-resistant infections reported during the same period that was mainly driven by an outbreak-causing, carbapenemase-producing Klebsiella pneumoniae (KPC) ST-11 strain. Genome-resolved metagenomics of urban waters before and after this outbreak, coupled with high-resolution whole-genome sequencing, confirmed the dissemination of the ST-11 strain and a novel KPC megaplasmid from the hospital to the urban environment. City-wide analysis showed that geospatial dissemination of the KPC megaplasmid in the urban environment inversely depended on the sewage system infrastructure.ConclusionsWe show how urban metagenomics and outbreak genomic surveillance can be coupled to generate relevant information for infection control, antibiotic stewardship, and pathogen epidemiology. Our results highlight the need to better characterize and understand how human-derived bacteria and antimicrobial resistance disseminate in the urban environment to incorporate this information in the development of effluent treatment infrastructure and public health policies.2A5ZQFmXmKxDXginhFNeyTVideo

Genomic investigation of clinically significant coagulase-negative staphylococci.

Introduction. Coagulase-negative staphylococci have been recognized both as emerging pathogens and contaminants of clinical samples. High-resolution genomic investigation may provide insights into their clinical significance.Aims. To review the literature regarding coagulase-negative staphylococcal infection and the utility of genomic methods to aid diagnosis and management, and to identify promising areas for future research.Methodology. We searched Google Scholar with the terms (Staphylococcus) AND (sequencing OR (infection)). We prioritized papers that addressed coagulase-negative staphylococci, genomic analysis, or infection.Results. A number of studies have investigated specimen-related, phenotypic and genetic factors associated with colonization, infection and virulence, but diagnosis remains problematic.Conclusion. Genomic investigation provides insights into the genetic diversity and natural history of colonization and infection. Such information allows the development of new methodologies to identify and compare relatedness and predict antimicrobial resistance. Future clinical studies that employ suitable sampling frames coupled with the application of high-resolution whole-genome sequencing may aid the development of more discriminatory diagnostic approaches to coagulase-staphylococcal infection.

Whole genome sequencing of Clostridioides difficile PCR ribotype 046 suggests transmission between pigs and humans.

A zoonotic association has been suggested for several PCR ribotypes (RTs) of Clostridioides difficile. In central parts of Sweden, RT046 was found dominant in neonatal pigs at the same time as a RT046 hospital C. difficile infection (CDI) outbreak occurred in the southern parts of the country. To detect possible transmission of RT046 between pig farms and human CDI cases in Sweden and investigate the diversity of RT046 in the pig population using whole genome sequencing (WGS). WGS was performed on 47 C. difficile isolates from pigs (n = 22), the farm environment (n = 7) and human cases of CDI (n = 18). Two different core genome multilocus sequencing typing (cgMLST) schemes were used together with a single nucleotide polymorphisms (SNP) analysis and the results were related to time and location of isolation of the isolates. The pig isolates were closely related (≤6 cgMLST alleles differing in both cgMLST schemes) and conserved over time and were clearly separated from isolates from the human hospital outbreak (≥76 and ≥90 cgMLST alleles differing in the two cgMLST schemes). However, two human isolates were closely related to the pig isolates, suggesting possible transmission. The SNP analysis was not more discriminate than cgMLST. No general pattern suggesting zoonotic transmission was apparent between pigs and humans, although contrasting results from two isolates still make transmission possible. Our results support the need for high resolution WGS typing when investigating hospital and environmental transmission of C. difficile.

Melioidosis fatalities in captive slender-tailed meerkats (Suricata suricatta): combining epidemiology, pathology and whole-genome sequencing supports variable mechanisms of transmission with one health implications

BackgroundMelioidosis is a tropical infectious disease which is being increasingly recognised throughout the globe. Infection occurs in humans and animals, typically through direct exposure to soil or water containing the environmental bacterium Burkholderia pseudomallei. Case clusters of melioidosis have been described in humans following severe weather events and in exotic animals imported into melioidosis endemic zones. Direct transmission of B. pseudomallei between animals and/or humans has been documented but is considered extremely rare. Between March 2015 and October 2016 eight fatal cases of melioidosis were reported in slender-tailed meerkats (Suricata suricatta) on display at a Wildlife Park in Northern Australia. To further investigate the melioidosis case cluster we sampled the meerkat enclosure and adjacent park areas and performed whole-genome sequencing (WGS) on all culture-positive B. pseudomallei environmental and clinical isolates.ResultsWGS confirmed that the fatalities were caused by two different B. pseudomallei sequence types (STs) but that seven of the meerkat isolates were highly similar on the whole-genome level. Used concurrently with detailed pathology data, our results demonstrate that the seven cases originated from a single original source, but routes of infection varied amongst meerkats belonging to the clonal outbreak cluster. Moreover, in some instances direct transmission may have transpired through wounds inflicted while fighting.ConclusionsCollectively, this study supports the use of high-resolution WGS to enhance epidemiological investigations into transmission modalities and pathogenesis of melioidosis, especially in the instance of a possible clonal outbreak scenario in exotic zoological collections. Such findings from an animal outbreak have important One Health implications.

Bacterial genome sequencing and analysis: paving the way for a Switzerland-wide molecular epidemiological surveillance platform.

Facing multidrug resistant (MDR) bacterial pathogens is one of the most important challenges for our society. The spread of highly virulent and resistant pathogens can be described using molecular typing technologies; in particular, whole genome sequencing (WGS) data can be usedf or molecular typing purposes with high resolution. WGS data analysis can explain the spatiotemporal patterns of pathogen transmission. However, the transmission between compartments (human, animal, food, environment) is very complex. Interoperable and curated metadata are a key requirement for fully understanding this complexity. In addition, high quality sequence data are a key element between centres using WGS data for diagnostic and epidemiological applications. We aim to describe steps to improve WGS data analysis and to implement a molecular surveillance platform allowing integration of high resolution WGS typing data and epidemiological data.

Improving the quality and workflow of bacterial genome sequencing and analysis: paving the way for a Switzerland-wide molecular epidemiological surveillance platform.

Facing multidrug resistant (MDR) bacterial pathogens is one of the most important challenges for our society. The spread of highly virulent and resistant pathogens can be described using molecular typing technologies; in particular, whole genome sequencing (WGS) data can be used for molecular typing purposes with high resolution. WGS data analysis can explain the spatiotemporal patterns of pathogen transmission. However, the transmission between compartments (human, animal, food, environment) is very complex. Interoperable and curated metadata are a key requirement for fully understanding this complexity. In addition, high quality sequence data are a key element between centres using WGS data for diagnostic and epidemiological applications. We aim to describe steps to improve WGS data analysis and to implement a molecular surveillance platform allowing integration of high resolution WGS typing data and epidemiological data.

1212. Whole Genome Sequencing for High-Resolution Methicillin-Resistant Staphylococcus aureus Outbreaks Tracing in Neonatal Intensive Care Units and In silico Resistance and Virulence Markers Detection

BackgroundThe French National Reference Center for Staphylococci used whole genome sequencing (WGS) to investigate outbreaks due to a virulent MRSA clone containing the toxic shock syndrome toxin-1 (TSST-1+, sequence type 5, Geraldine clone) increasingly reported in neonatal intensive care units (ICUs).MethodsWe analyzed 48 isolates previously characterized by spa typing: 31 isolates from outbreak 1 (infected or colonized patients, healthcare workers carriage and environment), 12 isolates from four distinct outbreaks (2, 3, 4, and 5) that occurred in geographically independent neonatal ICUs, and five sporadic strains. We performed WGS using a de novo assembly approach to perform comparisons between isolates (EpiSeq®, bioMérieux). A phylogenetic analysis was constructed by comparing single nucleotide variations (SNVs) in 2020 core-genes using a cutoff of 40 SNVs for defining isolates belonging to the same transmission cluster. We detected in silico resistance and virulence markers using the same bioinformatic pipeline.ResultsFor outbreak 1, 25/31 isolates with two distinct but related spa types t002 and t111 were highly related (<13 SNVs), suggesting the transmission of the same strain; 6/31 isolates were genetically distinct (>80 SNVs) from the previous cluster of 25 isolates suggesting their origin from separate sources. Interestingly the three isolates of outbreak 2 with a spa t111 differed by less than 22 SNVs from the main cluster of the 25 isolates of outbreak 1. This suggested origin from the same transmission cluster. The other three outbreaks showing respectively a spa t002 for outbreak 3 and outbreak 4 and a spa t045 for outbreak 5 were not affiliated to the main cluster of outbreak 1. The isolates carry numerous virulence factors (including TSST-1) and resistance markers confering a peculiar antibiotic resistance profile to the Geraldine clone.ConclusionWGS provides the resolution power to reveal unsuspected transmission events not indicated by conventional methods (different spa type). Based on its high resolution WGS is an all in one tool for epidemiology, virulence and resistance analysis. It really transforms outbreak management and infection control practice for an early response and should replace conventional methods for detection of MRSA transmission.Disclosures G. Durand, bioMérieux: Employee, Salary. F. Javerliat, bioMérieux: Employee, Salary.

Abstract 2460: Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma(MPM) is an aggressive cancer, which incidence has constantly increased over the past two decades and is expected to peak in 2020. The overall prognosis is poor, and predictive biomarkers of drug activity are missing. Given the heterogeneous and complex nature of MPM, it is likely that genomic aberrations changing the expression of several genes, might affect therapeutic response. Therefore, the aim of the present study was to identify genes whose copy number alterations might predict the MPM prognosis. Recurrent copy number alterations of genes were analyzed by high-resolution whole-genome sequencing in DNA obtained from a “discovery cohort” of 26 resected MPM patients treated with pemetrexed-based chemotherapy (8 with progressive disease, vs. 10 with stable disease and 8 with partial response). Prognostic markers identified by Copy Number Variation analysis with Nexus, Control-FREEC and ReadDepth software were validated by PCR gene copy number and gene expression analyses both in the “discovery” and in two “validation” cohorts of pemetrexed-treated and untreated patients (N=45 and 40). The role of emerging genes was evaluated through siRNA and pharmacological studies using proliferation, migration and apoptosis assays in MPM cells. As reported previously we observed copy number loss of CDKN2A (15.4%) and BAP1 (7.7%). Interestingly, copy number gain of NOTCH2 was observed in 50% of samples of the patients who underwent progression, whereas losses of PTP4A3 were associated with clinical benefit (SD+PR). The prognostic relevance of NOTCH2 was confirmed by PCR analysis. NOTCH-2 silencing reduced MPM cell migration and enhanced apoptosis induction by pemetrexed, while a PTP4A3 inhibitor overcame pemetrexed resistance in MPM cells characterized by high NOTCH2 and PTP4A3 expression. These results support the role of NOTCH2 as a novel prognostic/predictive biomarker for MPM, prompting prospective randomized trials for its validation. Moreover, preclinical data suggest that NOTCH2 and PTP4A3 are oncogenes suitable for effective therapeutic targeting in pemetrexed-resistant MPM cells. Citation Format: Leticia G. Leon, Maria Gemelli, Paolo A. Zucali, Christos Zoumadakis, Elisa Giovannetti. Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2017-2460

Context Is Everything: Harmonization of Critical Food Microbiology Descriptors and Metadata for Improved Food Safety and Surveillance.

Globalization of food networks increases opportunities for the spread of foodborne pathogens beyond borders and jurisdictions. High resolution whole-genome sequencing (WGS) subtyping of pathogens promises to vastly improve our ability to track and control foodborne disease, but to do so it must be combined with epidemiological, clinical, laboratory and other health care data (called “contextual data”) to be meaningfully interpreted for regulatory and health interventions, outbreak investigation, and risk assessment. However, current multi-jurisdictional pathogen surveillance and investigation efforts are complicated by time-consuming data re-entry, curation and integration of contextual information owing to a lack of interoperable standards and inconsistent reporting. A solution to these challenges is the use of ‘ontologies’ - hierarchies of well-defined and standardized vocabularies interconnected by logical relationships. Terms are specified by universal IDs enabling integration into highly regulated areas and multi-sector sharing (e.g., food and water microbiology with the veterinary sector). Institution-specific terms can be mapped to a given standard at different levels of granularity, maximizing comparability of contextual information according to jurisdictional policies. Fit-for-purpose ontologies provide contextual information with the auditability required for food safety laboratory accreditation. Our research efforts include the development of a Genomic Epidemiology Ontology (GenEpiO), and Food Ontology (FoodOn) that harmonize important laboratory, clinical and epidemiological data fields, as well as existing food resources. These efforts are supported by a global consortium of researchers and stakeholders worldwide. Since foodborne diseases do not respect international borders, uptake of such vocabularies will be crucial for multi-jurisdictional interpretation of WGS results and data sharing.

Genomic Investigations unmask Mycoplasma amphoriforme, a new respiratory pathogen.

Mycoplasma amphoriforme has been associated with infection in patients with primary antibody deficiency (PAD). Little is known about the natural history of infection with this organism and its ability to be transmitted in the community. The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative polymerase chain reaction. The genomes of all available isolates, originating from patients in the United Kingdom, France, and Tunisia, were sequenced along with the type strain. Genomic data were assembled and annotated, and a high-resolution phylogenetic tree was constructed. By using high-resolution whole-genome sequencing (WGS) data, we show that patients can be chronically infected with M. amphoriforme manifesting as a relapsing-remitting bacterial load, interspersed by periods when the organism is undetectable. Importantly, we demonstrate transmission of strains within a clinical environment. Antibiotic resistance mutations accumulate in isolates taken from patients who received multiple courses of antibiotics. Mycoplasma amphoriforme isolates form a closely related species responsible for a chronic relapsing and remitting infection in PAD patients in the United Kingdom and from immunocompetent patients in other countries. We provide strong evidence of transmission between patients attending the same clinic, suggesting that screening and isolation may be necessary for susceptible patients. This work demonstrates the critical role that WGS can play in rapidly unraveling the biology of a novel pathogen.

Whole Genome Sequencing Reveals Complex Evolution Patterns of Multidrug-Resistant Mycobacterium tuberculosis Beijing Strains in Patients

Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.

Genome-wide screen identifies new candidate genes associated with artemisinin susceptibility in Plasmodium falciparum in Kenya

Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-genome sequencing data in 27 isolates of Plasmodium falciparum. We identified genetic variants associated with susceptibility to dihydroartemisinin that implicate one region on chromosome 13, a candidate gene on chromosome 1 (PFA0220w, a UBP1 ortholog) and others (PFB0560w, PFB0630c, PFF0445w) with putative roles in protein homeostasis and stress response. There was a strong signal for positive selection on PFA0220w, but not the other candidate loci. Our results demonstrate the power of full-genome sequencing-based association studies for uncovering candidate genes that determine parasite sensitivity to artemisinins. Our study provides a unique reference for the interpretation of results from resistant infections.

An MBoC Favorite: High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli

Molecular Biology of the CellVol. 23, No. 11 MBoC 20th Anniversary FavoritesFree AccessAn MBoC Favorite: High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoliSui HuangSui HuangSearch for more papers by this authorPublished Online:13 Oct 2017https://doi.org/10.1091/mbc.e12-02-0155AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail In celebration of MBoC's first 20 years, members of the Editorial Board, members of the ASCB Council, and others comment on their favorite MBoC papers from the past two decades.This article is my favorite MBoC paper, because I believe that it broke ground for a new field—the study of the role of nucleoli in genome organization. Van Koningsbruggen et al. (2010) demonstrate that parts of all chromosomes are associated with nucleoli in human cells. The association is biased toward more transcriptionally repressive and AT-rich regions of the genome. The patterns of nucleolar-associated chromatins partially overlap with nuclear lamina–associated chromatin domains and are heritable through cell cycles. Nucleoli and nuclear lamina have long been known to nucleate heterochromatin. However, little attention has been directed to exploring possible roles of nucleoli in global genome organization. This article, together with a study published in the same period by Nemeth et al. (2010), provided the first molecular evidence of the extensive interactions between nucleoli and the genome. These findings raise the possibility that nucleoli play a key role in anchoring and organizing the mammalian genome.FOOTNOTESDOI: 10.1091/mbc.E12-02-0155Molecular Biology of the Cell Volume 23 Page 2016.REFERENCES Nemeth A, Conesa A, Santoyo-Lopez J, Medina I, Montaner D, Peterfia B, Solovei I, Cremer T, Dopazo J, Langst G (2010). Initial genomics of the human nucleolus. PLoS Genet 6, e1000889. Crossref, Medline, Google Scholar van Koningsbruggen S, Gierlinski M, Schofield P, Martin D, Barton GJ, Ariyurek Y, den Dunnen JT, Lamond AI (2010). High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli. Mol Biol Cell 21, 3735-3748. Link, Google ScholarFiguresReferencesRelatedDetailsCited byCalreticulin promotes EGF-induced EMT in pancreatic cancer cells via Integrin/EGFR-ERK/MAPK signaling pathway26 October 2017 | Cell Death & Disease, Vol. 8, No. 10 Vol. 23, No. 11 June 01, 20122015-2234 Metrics Downloads & Citations Downloads: 27Citations: 1 History Information© 2012 Huang. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).PDF download

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.