STATISTICAL ISSUES IN NEXT-GENERATION SEQUENCING

High throughput deep-sequencing or next-generation sequencing has emerged as an exciting new tool in a great number of applications (e.g., variant discovery, profiling of histone modifications, identifying transcription factor binding sites, resequencing, and transcriptome characterization). Even though this technology has generated unprecedented amounts of data in the scientific community few studies have looked carefully at its inherent variability. Recent studies of mRNA expression levels found little appreciable technical variation in Illumina’s Solexa sequencing platform (a next-generation sequencing device). Although these results are encouraging, they are limited to a specific platform and application, and have been made without any attention to experimental design. This paper provides an overview of some key issues in data management and experimental design related to Illumina’s Solexa Genome Analyzer technology

FUNCTIONAL DIVERGENCE OF DUPLICATED GENES IN THE SOYBEAN GENOME

The soybean genome has undergone many different evolutionary changes that are observable with modern technologies. Of particular interest to scientists and plant breeders is the fact that the soybean genome exhibits features of genome duplication from millions of years ago. Genes that were copied during the duplication event have since diverged functionally. Identifying functionally divergent duplicate genes may provide insight into the evolution of soybean. To investigate functional divergence, transcripts from seven different tissue samples of pooled soybean messenger RNA were sequenced using the Solexa next-generation sequencer and analyzed for gene expression. We tested differential expression of duplicated genes within tissue by employing an integrated normalization and statistical testing methodology. Blocks of duplicate genes (i.e., gene sets) were tested for unanimity of over-or under-expression. These same genes were also analyzed for differential expression across tissues. We identified thousands of duplicate genes that displayed differential expression patterns within each tissue. In some cases these genes were over-represented in duplicate blocks, suggestive of functional divergence of a large genomic region

α-Adrenergic inhibition of proliferation in HepG2 cells stably transfected with the α1B-adrenergic receptor through a p42MAP kinase/p21Cip1/WAF1-dependent pathway

AbstractActivation of α1B adrenergic receptors (α1BAR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of α1BAR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express α1BAR, were stably transfected with a rat α1BAR cDNA (TFG2 cells), in order to study the effects of maintained α1BAR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [3H]thymidine incorporation into DNA. Stimulation of α1BAR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G0/G1 and G2/M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42MAP kinase activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21Cip1/WAF1. Treatment of TFG2 cells with the specific MEK1 inhibitor PD98059, or infection with a −/− MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [3H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 −/− blunted the ability of phenylephrine to increase p21Cip1/WAF1 expression. In agreement with a role for increased p21Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of α1BARs in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via α1BAR appears to be p42MAP kinase and p21Cip1/WAF1 dependent

The Search for Supernova-produced Radionuclides in Terrestrial Deep-sea Archives

An enhanced concentration of 60Fe was found in a deep ocean's crust in 2004 in a layer corresponding to an age of ~2 Myr. The confirmation of this signal in terrestrial archives as supernova-induced and detection of other supernova-produced radionuclides is of great interest. We have identified two suitable marine sediment cores from the South Australian Basin and estimated the intensity of a possible signal of the supernova-produced radionuclides 26Al, 53Mn, 60Fe and the pure r-process element 244Pu in these cores. A finding of these radionuclides in a sediment core might allow to improve the time resolution of the signal and thus to link the signal to a supernova event in the solar vicinity ~2 Myr ago. Furthermore, it gives an insight on nucleosynthesis scenarios in massive stars, the condensation into dust grains and transport mechanisms from the supernova shell into the solar system

Integrative analysis of sequencing and array genotype data for discovering disease associations with rare mutations

High-throughput DNA sequencing provides an unprecedented opportunity to discover rare genetic variants associated with complex diseases and traits. However, sequencing a large number of subjects is prohibitively expensive. It is common to select subjects for sequencing from the cohorts that have collected genotyping array data. We impute the sequencing data from the array data for the cohort members who are not selected for sequencing and perform gene-level association tests for rare variants by properly combining the observed genotypes for sequenced subjects and the imputed genotypes for nonsequenced subjects. This integrative analysis is substantially more powerful than the use of sequencing data alone and can accelerate the search for disease-causing mutations

Imaging Changes after Stereotactic Radiosurgery of Primary and Secondary Malignant Brain Tumors

After radiosurgery of malignant tumors, it can be difficult to discriminate between transient treatment effects, radiation necrosis, and tumor progression on post-treatment imaging. Misinterpretation of an enlarging lesion may lead to inappropriate treatment and contribute to disagreements about treatment efficacy. In an effort to clarify this problem, we reviewed our experience with interpreting post-radiosurgical imaging in patients with malignant primary and secondary brain tumors. We reviewed results of radiosurgery of 30 malignant gliomas and 35 metastatic brain tumors with minimum follow up of 1 year or until death. Of 30 gliomas, 73% were larger a mean of 13 weeks after radiosurgery. Of 35 metatstatic tumors, 22% were larger a mean of 10 weeks after radiosurgery. Eleven had 18 F-fluorodeoxyglucose-positron emission tomography (FDG-PET) of enlarging lesions. Eight showed increased activity with respect to brain; three decreased activity. Of the eight, six predicted incorrectly based upon the patients' subsequent courses (all alive, mean follow up of 27 months), and two correctly, with the patients dying from the imaged lesions 8 and 13 months later. Of the three with FDG uptake less than brain, one patient was alive with 32 weeks of follow up, and two patients died from the imaged lesion 13 and 21 months later. Radiographic enlargement after radiosurgery is common, especially for gliomas. Physicians caring for these patients should be aware of this phenomenon and be cautious in interpreting post-treatment images. MRI appearance may be useful for metastases. FDG-PET seems unreliable. Further evaluation of Tl-201 and HMPAO SPECT or MRS is warranted.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45392/1/11060_2004_Article_393674.pd

Genome-wide Association and Population Genetic Analysis of C-Reactive Protein in African American and Hispanic American Women

C-reactive protein (CRP) is a systemic inflammation marker that predicts future cardiovascular risk. CRP levels are higher in African Americans and Hispanic Americans than in European Americans, but the genetic determinants of CRP in these admixed United States minority populations are largely unknown. We performed genome-wide association studies (GWASs) of 8,280 African American (AA) and 3,548 Hispanic American (HA) postmenopausal women from the Women's Health Initiative SNP Health Association Resource. We discovered and validated a CRP-associated variant of triggering receptors expressed by myeloid cells 2 (TREM2) in chromosomal region 6p21 (p = 10−10). The TREM2 variant associated with higher CRP is common in Africa but rare in other ancestral populations. In AA women, the CRP region in 1q23 contained a strong admixture association signal (p = 10−17), which appears to be related to several independent CRP-associated alleles; the strongest of these is present only in African ancestral populations and is associated with higher CRP. Of the other genomic loci previously associated with CRP through GWASs of European populations, most loci (LEPR, IL1RN, IL6R, GCKR, NLRP3, HNF1A, HNF4A, and APOC1) showed consistent patterns of association with CRP in AA and HA women. In summary, we have identified a common TREM2 variant associated with CRP in United States minority populations. The genetic architecture underlying the CRP phenotype in AA women is complex and involves genetic variants shared across populations, as well as variants specific to populations of African descent

Protein-coding variants implicate novel genes related to lipid homeostasis contributing to body-fat distribution.

Body-fat distribution is a risk factor for adverse cardiovascular health consequences. We analyzed the association of body-fat distribution, assessed by waist-to-hip ratio adjusted for body mass index, with 228,985 predicted coding and splice site variants available on exome arrays in up to 344,369 individuals from five major ancestries (discovery) and 132,177 European-ancestry individuals (validation). We identified 15 common (minor allele frequency, MAF ≥5%) and nine low-frequency or rare (MAF <5%) coding novel variants. Pathway/gene set enrichment analyses identified lipid particle, adiponectin, abnormal white adipose tissue physiology and bone development and morphology as important contributors to fat distribution, while cross-trait associations highlight cardiometabolic traits. In functional follow-up analyses, specifically in Drosophila RNAi-knockdowns, we observed a significant increase in the total body triglyceride levels for two genes (DNAH10 and PLXND1). We implicate novel genes in fat distribution, stressing the importance of interrogating low-frequency and protein-coding variants

Genome-wide association of white blood cell counts in Hispanic/Latino Americans: the Hispanic Community Health Study/Study of Latinos

Circulating white blood cell (WBC) counts (neutrophils, monocytes, lymphocytes, eosinophils, basophils) differ by ethnicity. The genetic factors underlying basal WBC traits in Hispanics/Latinos are unknown. We performed a genome-wide association study of total WBC and differential counts in a large, ethnically diverse US population sample of Hispanics/Latinos ascertained by the Hispanic Community Health Study and Study of Latinos (HCHS/SOL). We demonstrate that several previously known WBC-associated genetic loci (e.g. the African Duffy antigen receptor for chemokines null variant for neutrophil count) are generalizable to WBC traits in Hispanics/Latinos. We identified and replicated common and rare germ-line variants at FLT3 (a gene often somatically mutated in leukemia) associated with monocyte count. The common FLT3 variant rs76428106 has a large allele frequency differential between African and non-African populations. We also identified several novel genetic loci involving or regulating hematopoietic transcription factors (CEBPE-SLC7A7, CEBPA and CRBN-TRNT1) associated with basophil count. The minor allele of the CEBPE variant associated with lower basophil count has been previously associated with Amerindian ancestry and higher risk of acute lymphoblastic leukemia in Hispanics. Together, these data suggest that germline genetic variation affecting transcriptional and signaling pathways that underlie WBC development and lineage specification can contribute to inter-individual as well as ethnic differences in peripheral blood cell counts (normal hematopoiesis) in addition to susceptibility to leukemia (malignant hematopoiesis)