RNAseq analysis of bronchial epithelial cells to identify COPD-associated genes and SNPs

Abstract Background There is a need for more powerful methods to identify low-effect SNPs that contribute to hereditary COPD pathogenesis. We hypothesized that SNPs contributing to COPD risk through cis-regulatory effects are enriched in genes comprised by bronchial epithelial cell (BEC) expression patterns associated with COPD. Methods To test this hypothesis, normal BEC specimens were obtained by bronchoscopy from 60 subjects: 30 subjects with COPD defined by spirometry (FEV1/FVC < 0.7, FEV1% < 80%), and 30 non-COPD controls. Targeted next generation sequencing was used to measure total and allele-specific expression of 35 genes in genome maintenance (GM) genes pathways linked to COPD pathogenesis, including seven TP53 and CEBP transcription factor family members. Shrinkage linear discriminant analysis (SLDA) was used to identify COPD-classification models. COPD GWAS were queried for putative cis-regulatory SNPs in the targeted genes. Results On a network basis, TP53 and CEBP transcription factor pathway gene pair network connections, including key DNA repair gene ERCC5, were significantly different in COPD subjects (e.g., Wilcoxon rank sum test for closeness, p-value = 5.0E-11). ERCC5 SNP rs4150275 association with chronic bronchitis was identified in a set of Lung Health Study (LHS) COPD GWAS SNPs restricted to those in putative regulatory regions within the targeted genes, and this association was validated in the COPDgene non-hispanic white (NHW) GWAS. ERCC5 SNP rs4150275 is linked (D’ = 1) to ERCC5 SNP rs17655 which displayed differential allelic expression (DAE) in BEC and is an expression quantitative trait locus (eQTL) in lung tissue (p = 3.2E-7). SNPs in linkage (D’ = 1) with rs17655 were predicted to alter miRNA binding (rs873601). A classifier model that comprised gene features CAT, CEBPG, GPX1, KEAP1, TP73, and XPA had pooled 10-fold cross-validation receiver operator characteristic area under the curve of 75.4% (95% CI: 66.3%–89.3%). The prevalence of DAE was higher than expected (p = 0.0023) in the classifier genes. Conclusions GM genes comprised by COPD-associated BEC expression patterns were enriched for SNPs with cis-regulatory function, including a putative cis-rSNP in ERCC5 that was associated with COPD risk. These findings support additional total and allele-specific expression analysis of gene pathways with high prior likelihood for involvement in COPD pathogenesis.https://deepblue.lib.umich.edu/bitstream/2027.42/142723/1/12890_2018_Article_603.pd

Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing

Background: Clinical implementation of Next-Generation Sequencing (NGS) is challenged by poor control for stochastic sampling, library preparation biases and qualitative sequencing error. To address these challenges we developed and tested two hypotheses. Methods: Hypothesis 1: Analytical variation in quantification is predicted by stochastic sampling effects at input of (a) amplifiable nucleic acid target molecules into the library preparation, (b) amplicons from library into sequencer, or (c) both. We derived equations using Monte Carlo simulation to predict assay coefficient of variation (CV) based on these three working models and tested them against NGS data from specimens with well characterized molecule inputs and sequence counts prepared using competitive multiplex-PCR amplicon-based NGS library preparation method comprising synthetic internal standards (IS). Hypothesis 2: Frequencies of technically-derived qualitative sequencing errors (i.e., base substitution, insertion and deletion) observed at each base position in each target native template (NT) are concordant with those observed in respective competitive synthetic IS present in the same reaction. We measured error frequencies at each base position within amplicons from each of 30 target NT, then tested whether they correspond to those within the 30 respective IS. Results: For hypothesis 1, the Monte Carlo model derived from both sampling events best predicted CV and explained 74% of observed assay variance. For hypothesis 2, observed frequency and type of sequence variation at each base position within each IS was concordant with that observed in respective NTs (R2 = 0.93). Conclusion: In targeted NGS, synthetic competitive IS control for stochastic sampling at input of both target into library preparation and of target library product into sequencer, and control for qualitative errors generated during library preparation and sequencing. These controls enable accurate clinical diagnostic reporting of confidence limits and limit of detection for copy number measurement, and of frequency for each actionable mutation

Genome maintenance gene regulation in normal bronchial epithelial cells by CEBP and TP53 transcription factor families is different in COPD subjects

Rationale: Transcription factors in the CEBP and TP53 families regulate key antioxidant (AO), DNA repair (DNAR), and cell cycle control (CCC) genes in normal bronchial epithelial cells (NBEC). In turn, these genome maintenance genes are important for protecting NBEC from damage, and sub-optimal function of these gene pathways is associated with risk for COPD. In this study, we investigated expression patterns of CEBP and TP53 transcription factor family gene pathways in NBEC of COPD and non-COPD subjects in an effort to identify a biomarker for COPD risk and to better understand mechanisms of COPD risk. Methods: NBEC samples were obtained by bronchoscopy from 30 COPD subjects defined by spirometry (FEV1/FVC \u3c0.7), and 30 non-COPD controls. A competitive multiplex PCR-amplicon method was used to generate targeted libraries for next generation sequencing (NGS) analysis of the transcription factors CEBPA, CEBPG, CEBPD, TP53, TP63, and TP73 transcription factors, and 24 AO, DNAR, and CCC genes that are known or putative targets for these transcription factors. Shrinkage Linear Discriminant Analysis (SLDA) and 10-fold cross-validation was used to identify a model COPD classifier. Results: The model with the best AUC ROC comprised eight gene targets, including KEAP1, XPA, GPX1, TP73, CAT, CEBPG, TP53, CEBPD, with pooled 10-fold cross-validation AUC of 76.3% (95% CI: 72%-93%). To further explore the regulatory relationships underlying this model, we assessed inter-gene correlation difference in COPD by Fisher Z-test, network of correlation analysis, ANCOVA and multivariate linear regression. CEBPD was expressed higher in NBEC of COPD compared to controls. Gene-pairs negatively or non-correlated with CEBPD in controls were positively correlated in COPD (p-value of Z-score \u3c0.05). Further, CEBPA expression was more disperse in COPD compared to non-COPD control (p-value =0.02). TP53 was positively correlated with CDKN1A, ERCC4 and TFE3 in controls and correlation of each gene-pair was significantly lower among COPD subjects (p-value of Z-score \u3c0.05). Further, KEAP1, which is regulated by TP53, was expressed lower in NBEC of COPD compared to controls. In contrast, TP63 and CDKN1A were negatively correlated in controls and positively correlated in COPD (p-value = 0.002). Conclusion: CEBP and TP53 transcription factor family regulation of many AO, DNAR, and CCC pathway genes in NBEC was different in COPD subjects compared with non-COPD controls. A classifier based on these differences may enable more accurate identification of individuals at highest risk for COPD

Haplotype and diplotype analyses of variation in ERCC5 transcription cis-regulation in normal bronchial epithelial cells

Excision repair cross-complementation group 5 (ERCC5) gene plays an important role in nucleotide excision repair, and dysregulation of ERCC5 is associated with increased lung cancer risk. Haplotype and diplotype analyses were conducted in normal bronchial epithelial cells (NBEC) to better understand mechanisms responsible for interindividual variation in transcript abundance regulation of ERCC5 We determined genotypes at putative ERCC5 cis-regulatory SNPs (cis-rSNP) rs751402 and rs2296147, and marker SNPs rs1047768 and rs17655. ERCC5 allele-specific transcript abundance was assessed by a recently developed targeted sequencing method. Syntenic relationships among alleles at rs751402, rs2296147, and rs1047768 were assessed by allele-specific PCR followed by Sanger sequencing. We then assessed association of ERCC5 allele-specific expression at rs1047768 with haplotype and diplotype structure at cis-rSNPs rs751402 and rs2296147. Genotype analysis revealed significantly (P \u3c 0.005) higher interindividual variation in allelic ratios in cDNA samples relative to matched gDNA samples at both rs1047768 and rs17655. By diplotype analysis, mean expression was higher at the rs1047768 alleles syntenic with rs2296147 T allele compared with rs2296147 C allele. Furthermore, mean expression was lower at rs17655 C allele, which is syntenic with G allele at a linked SNP rs873601 (D\u27 = 0.95). These data support the conclusions that in NBEC, T allele at SNP rs2296147 upregulates ERCC5, variation at rs751402 does not alter ERCC5 regulation, and that C allele at SNP rs17655 downregulates ERCC5 Variation in ERCC5 transcript abundance associated with allelic variation at these SNPs could result in variation in NER function in NBEC and lung cancer risk

Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics

Additional file 1: Table S1. of A lung cancer risk classifier comprising genome maintenance genes measured in normal bronchial epithelial cells

List of assays, SNP site, primer and internal standard sequences, demographics, and transcript abundance (target gene molecules/106 ACTB molecules). Bold: Selected features. (XLSX 103 kb