O. Colin Stine

Differential abundance analysis for microbial marker-gene surveys

We introduce a methodology to assess differential abundance in sparse high-throughput microbial marker-gene survey data. Our approach, implemented in the metagenomeSeq Bioconductor package, relies on a novel normalization technique and a statistical model that accounts for undersampling-a common feature of large-scale marker-gene studies. Using simulated data and several published microbiota data sets, we show that metagenomeSeq outperforms the tools currently used in this field.We introduce a methodology to assess differential abundance in sparse high-throughput microbial marker-gene survey data. Our approach, implemented in the metagenomeSeq Bioconductor package, relies on a novel normalization technique and a statistical model that accounts for undersampling—a common feature of large-scale marker-gene studies. Using simulated data and several published microbiota data sets, we show that metagenomeSeq outperforms the tools currently used in this field.

Genome Sequence of Aeromonas hydrophila ATCC 7966T: Jack of All Trades

The complete genome of Aeromonas hydrophila ATCC 7966(T) was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966(T). Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments.

Initial genome scan of the nimh genetics initiative bipolar pedigrees: Chromosomes 1, 6, 8, 10, and 12

A report on an initial genome screen on 540 individuals in 97 families was collected as part of the NIMH Genetics Initiative on Bipolar Disorder. Families were ascertained to be informative for genetic linkage and underwent a common ascertainment and assessment protocol at four clinical sites. The sample was genotyped for 65 highly polymorphic markers from chromosomes 1, 6, 8, 10, and 12. The average intermarker interval was 16 cM. Genotypic data was analyzed using affected sib pair, multipoint affected sib pair, and pedigree analysis methods. Multipoint methods gave lod scores of approximately two on chromosomes 1, 6, and 10. The peak lod score on chromosome 6 occurred at the end of the q-arm, at some distance from the 6p24-22 area previously implicated for schizophrenia. We are currently genotyping additional markers to reduce the intermarker interval around the signals. The interpretation of results from a genome screen of a complex disorder and the problem of achieving a balance between detecting false positive results and the ability to detect genes of modest effect are discussed.

Deep sequencing of the oral microbiome reveals signatures of periodontal disease.

The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (∼2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ∼90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.

Major Genes Regulating Total Serum Immunoglobulin E Levels in Families with Asthma

Immunoglobulin E (IgE) has a major role in the pathogenesis of allergic disorders and asthma. Previous data from 92 families, each identified through a proband with asthma, showed evidence for two major genes regulating total serum IgE levels. One of these genes mapped to 5q31-33. In the current study, the segregation analysis was extended by the addition of 108 probands and their families, ascertained in the same manner. A mixed recessive model (i.e., major recessive gene and residual genetic effect) was the best-fitting and most-parsimonious one-locus model of the segregation analysis. A mixed two-major-gene model (i.e., two major genes and residual genetic effect) fit the data significantly better than did the mixed recessive one-major-gene model. The second gene modified the effect of the first recessive gene. Individuals with the genotype aaBB (homozygous high-risk allele at the first gene and homozygous low-risk allele at the second locus) had normal IgE levels (mean 23 IU/ml), and only individuals with genotypes aaBb and aabb had high IgE levels (mean 282 IU/ml). A genomewide screening was performed using variance-component analysis. Significant evidence for linkage was found for a novel locus at 7q, with a multipoint LOD score of 3. 36 (P=.00004). A LOD score of 3.65 (P=.00002) was obtained after genotyping additional markers in this region. Evidence for linkage was also found for two previously reported regions, 5q and 12q, with LOD scores of 2.73 (P=.0002) and 2.46 (P=.0004), respectively. These results suggest that several major genes, plus residual genetic effects, regulate total serum IgE levels.

Multilocus Sequence Typing for Characterization of Clinical and Environmental Salmonella Strains

ABSTRACT Multilocus sequence typing (MLST) based on the 16S RNA, pduF, glnA, and manB genes was developed for Salmonella, and its discriminatory ability was compared to those of pulsed-field gel electrophoresis (PFGE) and serotyping. PFGE differentiated several strains undifferentiable by serotyping, and 78 distinct PFGE types were identified among 231 Salmonella isolates grouped into 22 serotypes and 12 strains of undetermined serotype. The strains of several PFGE types were further differentiated by MLST, which suggests that the discriminatory ability of MLST for the typing of Salmonella is better than that of serotyping and/or PFGE typing. manB-based sequence typing identified two distinct genetic clusters containing 32 of 54 (59%) clinical isolates whose manB gene sequences were analyzed. The G+C contents and Splitstree analysis of the manB, glnA, and pduF genes of Salmonella indicated that the genes differ in their evolutionary origins and that recombination played a significant role in their evolution.

Initial genomic scan of the NIMH genetics initiative bipolar pedigrees: Chromosomes 3, 5, 15, 16, 17, and 22

As part of the four-center NIMH Genetics Initiative on Bipolar Disorder we carried out a genomic scan of chromosomes 3, 5, 15, 16,17, and 22. Genotyping was performed on a set of 540 DNAs from 97 families, enriched for affected relative pairs and parents where available. We report here the results of the initial 74 markers that have been typed on this set of DNAs. The average distance between markers (theta) was 12.3 cM. Nonparametric analysis of excess allele sharing among affected sibling pairs used the SIBPAL program of the S.A.G.E. package to test three hierarchical models of affected status. D16S2619 gave some evidence of linkage to bipolar disorder, with P = 0.006 for Model II (in which bipolar 1, bipolar 2 and schizoaffective-bipolar type individuals are considered affected). Nearby markers also showed increased allele sharing. A second interesting region was toward the telomere of chromosome 5q, where D5S1456 and nearby markers showed increased allele sharing; for D5S1456, P = 0.05, 0.015 and 0.008 as the models of affected status become more broad. MOD score analysis also supported the possible presence of a susceptibility locus in this region of chromosome 5. A pair of adjacent markers on chromosome 3, D3S2405 and D3S3038, showed a modest increased allele sharing in the broad model. Several isolated markers had excess allele sharing at the P < 0.05 level under a single model. D15S217 showed a MOD score of 2.37 (P < 0.025). Multipoint analysis flagged the region of chromosome 22 around D22S533 as the most interesting. Thus, several regions showed modest evidence for linkage to bipolar disorder in this initial genomic scan of these chromosomes, including broad regions near previous reports of possible linkage.

Genome-wide scan of bipolar disorder in 65 pedigrees: Supportive evidence for linkage at 8q24, 18q22, 4q32, 2p12, and 13q12

The purpose of this study was to assess 65 pedigrees ascertained through a Bipolar I (BPI) proband for evidence of linkage, using nonparametric methods in a genome-wide scan and for possible parent of origin effect using several analytical methods. We identified 15 loci with nominally significant evidence for increased allele sharing among affected relative pairs. Eight of these regions, at 8q24, 18q22, 4q32, 13q12, 4q35, 10q26, 2p12, and 12q24, directly overlap with previously reported evidence of linkage to bipolar disorder. Five regions at 20p13, 2p22, 14q23, 9p13, and 1q41 are within several Mb of previously reported regions. We report our findings in rank order and the top five markers had an NPL>2.5. The peak finding in these regions were D8S256 at 8q24, NPL 3.13; D18S878 at 18q22, NPL 2.90; D4S1629 at 4q32, NPL 2.80; D2S99 at 2p12, NPL 2.54; and D13S1493 at 13q12, NPL 2.53. No locus produced statistically significant evidence for linkage at the genome-wide level. The parent of origin effect was studied and consistent with our previous findings, evidence for a locus on 18q22 was predominantly from families wherein the father or paternal lineage was affected. There was evidence consistent with paternal imprinting at the loci on 13q12 and 1q41.

Multilocus Sequence Typing Reveals a Lack of Diversity among Escherichia coli O157:H7 Isolates That Are Distinct by Pulsed-Field Gel Electrophoresis

ABSTRACT Escherichia coli O157:H7 is a major cause of foodborne illness in the United States. Pulsed-field gel electrophoresis (PFGE) is the molecular epidemiologic method mostly commonly used to identify food-borne outbreaks. Although PFGE is a powerful epidemiologic tool, it has disadvantages that make a DNA sequence-based approach potentially attractive. Multilocus sequence typing (MLST) analyzes the internal fragments of housekeeping genes to establish genetic relatedness between isolates. We sequenced selected portions of seven housekeeping genes and two membrane protein genes (ompA and espA) of 77 isolates that were diverse by PFGE to determine whether there was sufficient sequence variation to be useful as an epidemiologic tool. There was no DNA sequence diversity in the sequenced portions of the seven housekeeping genes and espA. For ompA, all but five isolates had sequence identical to that of the reference strains. E. coli O157:H7 has a striking lack of genetic diversity in the genes we explored, even among isolates that are clearly distinct by PFGE. Other approaches to identify improved molecular subtyping methods for E. coli 0157:H7 are needed.

Genomic Survey of Bipolar Illness in the NIMH Genetics Initiative Pedigrees: A Preliminary Report

NIMH Genetics Initiative Bipolar Group: John I. Nurnberger, Jr.* (Chair), J. Raymond DePaulo,Elliot S. Gershon, Theodore Reich, Mary C. Blehar, and collaborators from Indiana University(Howard J. Edenberg, Tatiana Foroud, Marvin Miller, Elizabeth Bowman, Aimee Mayeda, N. LeelaRau, Carrie Smiley, and P. Michael Conneally), Johns Hopkins University (Francis Mc-Mahon, Deborah Meyers, Sylvia Simpson, Melvin McInnis, and O. Colin Stine), NIMH IntramuralResearch Program (Sevilla Detera-Wadleigh, Lynn Goldin, Juliet Guroff, Elizabeth Max-well, Diane Kazuba, Pablo V. Gejman, Judith Badner, and Alan Sanders), and WashingtonUniversity of St. Louis (John Rice, Laura Bierut, and Alison Goate).Four sites collaborated with the NIMH todevelop a resource for the genetic study ofbipolar (BP) illness. Common methods of as-certainment and assessment were devel-oped in 1989. A series of families with a bi-polar I (BPI) proband and at least one BPIor schizoaffective, bipolar type (SA/BP)first-degree relative has been studied. Wenow report initial data from a genomic sur-vey with an average intermarker interval of10 cM on 540 subjects from 97 families. Thisis the largest commonly ascertained and as-sessed linkage sample for bipolar illness re-ported to date; it includes 232 subjects withBPI, 32 SA/BP, 72 bipolar II (BPII), and 88unipolar, recurrent (UPR). Nonparametricmethods of analysis were employed, with allsites using affected sib pair analysis. Thestrongest findings thus far appear to be onchromosomes 1, 6, 7, 10, 16, and 22. Supporthas also been found for some previously re-ported linkages, including 21q and possiblyXq26. All these areas (as well as others) willbe followed up with additional markers andfurther analyses. No locus tested thus farmeets stringent criteria for an initial find-ing of significant linkage. Am. J. Med. Genet.74:227–237, 1997.