A computer simulation analysis of the accuracy of partial genome sequencing and restriction fragment analysis in estimating genetic relationships: an application to papillomavirus DNA sequences

BACKGROUND: Determination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny. However, there is little information available on how well the genetic relationships inferred from different genotyping methods agree with true genetic relationships. In this report, two genotyping methods – restriction fragment analysis (RFA) and partial genome DNA sequencing – were each compared to complete DNA sequencing as the definitive standard for classification. RESULTS: Using the Genbank database, 16 different types or subtypes of papillomavirus were selected as study samples, because numerous complete genome sequences were available. RFA was achieved by computer-simulated digestion. The genetic similarity of samples, based on RFA, was determined from the proportion of fragments that matched in size. DNA sequences of four specific genes (E1, E6, E7, and L1), representing partial genome sequencing, were also selected for comparison to complete genome sequencing. Laboratory error was not taken into account. Evaluation of the correlation between genetic similarity matrices (Mantel's r) and comparisons of the structure of the derived dendrograms (partition metric) indicated that partial genome sequencing (for single genes) had higher agreement with complete genome sequencing, achieving a maximum Mantel's r = 0.97 and a minimum partition metric = 10. RFA had lower agreement, with a maximum Mantel's r = 0.60 and a minimum partition metric = 18. CONCLUSIONS: This simulation indicated that for smaller genomes, such as papillomavirus, partial genome sequencing is superior to restriction fragment analysis in representing genetic relatedness among isolates. The generalizability of these results to larger genomes, as well as the impact of laboratory error, remains to be demonstrated

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7

Background: It is well known that P16INK4A, P14ARF, P15INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16INK4A expression is mediated with CBX7.Results: In this study, we found that the upregulation of P16INK4A, P14ARF, P15INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16INK4A, P14ARF and P15INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells.Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16INK4A, P14ARF, P15INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes’ expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters

Identification of patterns of transmission of Salmonella within swine production systems using pulsed field gel electrophoresis (PFGE) and repetitive sequence polymerase chain reaction (REP-PCR): a quantitative analysis

Pulsed field gel e lectrophoresis (PFGE) using 3 enzymes (Spe I, Xba I, Avr II) and repetitive sequence polymerase chain reaction (REP-PCR) with 3 primers (BOX, ERIC, REP) were compared with respect to their validity as a method for identifying transmission of Salmonella on swine farms. Sixty-eight isolates of Salmonella were obtained from feces of swine, cats, mice, and birds, insect body parts, water and floor samples, and boot scrapings collected on 9 swine farms in Illinois USA. Genetic distances between isolates were calculated using the Dice matching coefficient. Cluster analysis of distance matrices was conducted using the UPGMA algorithm

Ten-year cancer incidence in rescue/recovery workers and civilians exposed to the September 11, 2001 terrorist attacks on the World Trade Center

Background: Cancer incidence in exposed rescue/recovery workers (RRWs) and civilians (non-RRWs) was previously reported through 2008. Methods: We studied occurrence of first primary cancer among World Trade Center Health Registry enrollees through 2011 using adjusted standardized incidence ratios (SIRs), and the WTC-exposure-cancer association, using Cox proportional hazards models. Results: All-cancer SIR was 1.11 (95% confidence interval (CI) 1.03–1.20) in RRWs, and 1.08 (95% CI 1.02–1.15) in non-RRWs. Prostate cancer and skin melanoma were significantly elevated in both populations. Thyroid cancer was significantly elevated only in RRWs while breast cancer and non-Hodgkin's lymphoma were significantly elevated only in non-RRWs. There was a significant exposure dose-response for bladder cancer among RRWs, and for skin melanoma among non-RRWs. Conclusions: We observed excesses of total and specific cancers in both populations, although the strength of the evidence for causal relationships to WTC exposures is somewhat limited. Continued monitoring of this population is indicated

Investigations of the Accuracy of Partial Genome Sequencing and Restriction Fragment Analysis in Determination of Genetic Relationships: A Computer Simulation Study

184 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2003.A computer simulation approach has been used to evaluate the accuracy of partial genome sequencing and restriction fragment analysis in determination of genetic relationships, as represented in phylogenetic trees. Three partial genome DNA sequences with different lengths (200, 600, 1000 bps) and three analytic algorithms for restriction fragment data (the Dice coefficient, the Nei and Li distance, and the Weigel and Scherba distance) were considered. The effects of genetic factors (genome length, overall nucleotide substitution rate, and extent of rate variation among sites) and model tree parameters (extent of rate variation among lineages, tree imbalance, and tree stemminess) on accuracy were also evaluated. Thirty-two model trees, each with different values of the three tree parameters, were selected. For each model tree, genomic DNA sequences were simulated, with resultant variation in the three genetic factors. The generated extant DNA sequences were analyzed using partial genome sequencing and restriction fragment analysis, and the phylogenetic relationships among the DNA sequences were reconstructed. These phylogenetic trees were then compared with the model trees, and accuracy was evaluated for two different aspects: similarity of topology and similarity of branch lengths. The analysis indicated that partial genome sequencing generally was more accurate in phylogenetic reconstruction than restriction fragment analysis. However, many factors modified the relative accuracy of these two techniques. Longer partial genome sequences produced more accurate results. The three analytic algorithms for restriction fragment data had different accuracy, with the Dice coefficient and the Nei and Li distance differing little, with both more accurate than the Weigel and Scherba distance. The associations of genetic factors and model tree parameters with the accuracy of phylogenetic reconstruction were complicated and varied among techniques, analytic conditions, and the measure of accuracy used. Based on findings from this study, some recommendations regarding the utility of the two molecular techniques and the analytic algorithms, and the selection of gene or region for sequence analysis were provided.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Investigations of the Accuracy of Partial Genome Sequencing and Restriction Fragment Analysis in Determination of Genetic Relationships: A Computer Simulation Study

184 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2003.A computer simulation approach has been used to evaluate the accuracy of partial genome sequencing and restriction fragment analysis in determination of genetic relationships, as represented in phylogenetic trees. Three partial genome DNA sequences with different lengths (200, 600, 1000 bps) and three analytic algorithms for restriction fragment data (the Dice coefficient, the Nei and Li distance, and the Weigel and Scherba distance) were considered. The effects of genetic factors (genome length, overall nucleotide substitution rate, and extent of rate variation among sites) and model tree parameters (extent of rate variation among lineages, tree imbalance, and tree stemminess) on accuracy were also evaluated. Thirty-two model trees, each with different values of the three tree parameters, were selected. For each model tree, genomic DNA sequences were simulated, with resultant variation in the three genetic factors. The generated extant DNA sequences were analyzed using partial genome sequencing and restriction fragment analysis, and the phylogenetic relationships among the DNA sequences were reconstructed. These phylogenetic trees were then compared with the model trees, and accuracy was evaluated for two different aspects: similarity of topology and similarity of branch lengths. The analysis indicated that partial genome sequencing generally was more accurate in phylogenetic reconstruction than restriction fragment analysis. However, many factors modified the relative accuracy of these two techniques. Longer partial genome sequences produced more accurate results. The three analytic algorithms for restriction fragment data had different accuracy, with the Dice coefficient and the Nei and Li distance differing little, with both more accurate than the Weigel and Scherba distance. The associations of genetic factors and model tree parameters with the accuracy of phylogenetic reconstruction were complicated and varied among techniques, analytic conditions, and the measure of accuracy used. Based on findings from this study, some recommendations regarding the utility of the two molecular techniques and the analytic algorithms, and the selection of gene or region for sequence analysis were provided.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD