Jiyoun Yeo

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

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 worlds 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 (R2 = 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/106 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.

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.

A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

Background Reverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed. Methods We developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples. Results Reagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples. Conclusions This quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

Abstract 2890: ERCC5 variant rs2296147 T-allele creates a predicted TP53 binding site and up-regulates transcript abundance in normal bronchial epithelial cells, while rs17655 C-allele is linked to miRNA binding site variant and down regulates

Background: Excision repair cross-complementation group 5 (ERCC5) gene plays an important role in nucleotide excision repair (NER) and dysregulation of ERCC5 is associated with increased lung cancer risk. This study was conducted to characterize cis-acting genetic variants responsible for inter-individual variation in ERCC5 transcript regulation in normal bronchial epithelial cells (NBEC). Methods: We determined genotypes at putative ERCC5 cis-regulatory single nucleotide polymorphic sites (SNP) rs751402 and rs2296147, and marker SNPs rs1047768 and rs17655. Using a recently developed targeted sequencing method, ERCC5 allele-specific transcript abundance was assessed in NBEC RNA from 55 individuals heterozygous for rs1047768 and 21 subjects heterozygous for rs17655. Syntenic relationships among alleles at rs751402, rs2296147 and rs1047768 were assessed by allele-specific PCR followed by Sanger sequencing. We assessed association of NBEC ERCC5 allele-specific expression at rs1047768 with haplotype and diplotype structure at putative ERCC5 promoter cis-regulatory SNPs rs751402 and rs2296147. Results: Genotype analysis revealed higher inter-individual variation in allelic ratios in cDNA samples relative to matched gDNA samples at both rs1047768 and rs17655 (p Conclusions: These data support the conclusion that T allele at SNP rs2296147 creates a TP53 binding site and up-regulates ERCC5 while C allele at SNP rs873601 creates a miRNA binding site and down-regulates ERCC5. Variation in ERCC5 transcript abundance associated with allelic variation at these SNPs is likely associated with variation in NER function in NBEC and lung cancer risk. Citation Format: Xiaolu Zhang, Erin L. Crawford, Thomas M. Blomquist, Sadik A. Khuder, Jiyoun Yeo, Albert M. Levin, James C. Willey. ERCC5 variant rs2296147 T-allele creates a predicted TP53 binding site and up-regulates transcript abundance in normal bronchial epithelial cells, while rs17655 C-allele is linked to miRNA binding site variant and down regulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2890.

Disparate effects of antibiotics on hypertension

Gut microbiota are associated with a variety of complex polygenic diseases. The usage of broad-spectrum antibiotics by patients affected by such diseases is an important environmental factor to consider, because antibiotics, which are widely prescribed to curb pathological bacterial infections, also indiscriminately eliminate gut commensal microbiota. However, the extent to which antibiotics reshape gut microbiota and per se contribute to these complex diseases is understudied. Because genetics play an important role in predisposing individuals to these modern diseases, we hypothesize that the extent to which antibiotics influence complex diseases depends on the host genome and metagenome. The current study tests this hypothesis in the context of hypertension, which is a serious risk factor for cardiovascular diseases. A 3 × 2 factorial design was used to test the blood pressure (BP) and microbiotal effects of three different antibiotics, neomycin, minocycline, and vancomycin, on two well-known, preclinical, genetic models of hypertension, the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR), both of which develop hypertension, but for different genetic reasons. Regardless of the class, oral administration of antibiotics increased systolic blood pressure of the S rat, while minocycline and vancomycin, but not neomycin, lowered systolic blood pressure in the SHR. These disparate BP effects were accompanied by significant alterations in gut microbiota. Our study highlights the need to consider an individualized approach for the usage of antibiotics among hypertensives, as their BP could be affected differentially based on their individual genetic and microbiotal communities.

Abstract 3381: Investigation of C/EBPG transcription factor role in regulation of ERCC4 and ERCC5 in human lung cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: ERCC4 and ERCC5 are key nucleotide excision DNA repair genes and are expressed abundantly in normal bronchial epithelial cells (NBEC). DNA sequence variation in ERCC4 or ERCC5 is associated with risk for lung cancer in multiple independent studies. C/EBPG expression is correlated with that of ERCC4, ERCC5 and many other key genes in BEC, suggesting a regulatory role, supported by experimental observation that up-regulation of CCAAT/enhancer-binding protein gamma (C/EBPG) transcription factor up-regulates ERCC5 expression in H23 lung cancer cell line. The purpose of this study was to test the hypothesis that knockdown of C/EBPG in lung cancer cells would reduce transcription of ERCC5 and ERCC4. Methods: We knocked-down C/EBPG transcript level by C/EBPG siRNA transfection in three non-small cell lung cancer cell lines: H23, H520 and H1703. Following transfection, RNA was extracted after 24 and 48 hours. Reduction of C/EBPG transcript abundance measured by competitive multiplex RT-PCR in H23, H520, and H1703 was 56%, 84%, and 92% at 24 hours, and 82%, 89%, and 76% at 48 hours. After confirming C/EBPG knockdown, we measured allele-specific expression (ASE) and total expression of C/EBPG, ERCC4 ERCC5 through competitive multiplex PCR-based amplicon sequencing library preparation followed by Illumina HiSeq next generation sequencing (NGS) (Blomquist et al, PLOS one, 2013). This NGS method a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. ASE was measured at ERCC4 SNPs rs2276466, rs3743538 and ERCC5 SNPs rs1047768, rs17655, rs4150316. Results: In H23 C/EBPG knock-down was associated with no change in expression at any of the SNPs for ERCC4 or ERCC5, while in H520 one ERCC5 rs1047768 allele decreased (3-fold) and the other allele had no change, and in H1703 both ERCC5 rs1047768 alleles increased (10-fold, and 2-fold). In H520, ERCC4 measured at two SNPs were different with increase at one allele but not the other at rs3743538 and decrease for both alleles at rs2276466. In H1703 each allele at ERCC4 rs3743538 increased, 2-fold and 45-fold respectively and each allele at ERCC4 rs2276466 increased, 5-fold and 2.7-fold respectively. Conclusions: These results support prior evidence that C/EBPG regulates ERCC5 transcription, and provides evidence that it also contributes to regulation of ERCC4. The inter-allelic and inter-cell line variation in response to C/EBPG knockdown data supports that hypothesis that cis-regulatory DNA variants interact with C/EBPG in regulation of these genes. The lack of response in H23 may be in part due to the low constitutive level of ERCC4 and ERCC5 expression in this cell line. Citation Format: Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, James C. Willey. Investigation of C/EBPG transcription factor role in regulation of ERCC4 and ERCC5 in human lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3381. doi:10.1158/1538-7445.AM2014-3381

Salt-Responsive Metabolite, β-Hydroxybutyrate, Attenuates Hypertension

SUMMARY Dietary salt reduction and exercise are lifestyle modifications for salt-sensitive hypertensives. While exercise has prominent metabolic effects, salt has an adverse effect on metabolic syndrome, of which hypertension is a hallmark. We hypothesized that dietary salt impacts metabolism in a salt-sensitive model of hypertension. An untargeted metabolomic approach demonstrates lower circulating levels of the ketone body, beta-hydroxybutyrate (βOHB), in high salt-fed hypertensive rats. Despite the high salt intake, specific rescue of βOHB levels by nutritional supplementation of its precursor, 1,3-butanediol, attenuates hypertension and protects kidney function. This beneficial effect of βOHB was likely independent of gut-microbiotal and Th17-mediated effects of salt and instead facilitated by βOHB inhibiting the renal Nlrp3 inflammasome. The juxtaposed effects of dietary salt and exercise on salt-sensitive hypertension, which decrease and increase βOHB respectively, indicate that nutritional supplementation of a precursor of βOHB provides a similar benefit to salt-sensitive hypertension as exercise.

Abstract 1983: Altered regulation of CEBP transcription factor family in normal bronchial epithelial cells of subjects with lung cancer or COPD

Background: Antioxidant (AO), DNA repair (DNAR), and cell cycle control (CCC) genes play a role in protecting normal bronchial epithelial cells (NBEC) from damage, and sub-optimal function is associated with risk for COPD and lung cancer. CEBP transcription factors regulate key AO and DNA repair genes in NBEC. In this study, we investigated the association between CEBP transcription factor function and regulation of these gene pathways in NBEC of cancer (CA) and non-cancer (NC) COPD subjects. Methods: NBEC samples were obtained by bronchoscopy from 66 CA cases and 60 matched NC controls, and 30 NC COPD and 30 NC non-COPD controls defined by spirometry (FEV1 Citation Format: Jiyoun Yeo, Erin Crawford, Xiaolu Zhang, Albert Levin, James Willey. Altered regulation of CEBP transcription factor family in normal bronchial epithelial cells of subjects with lung cancer or COPD. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1983.

Abstract 2905: Induction of hTERT and increased proliferative potential in conditionally reprogrammed normal bronchial epithelial cells

Background. Based on increasing evidence from this laboratory and genome wide association studies (GWAS), single nucleotide polymorphisms (SNPs) responsible for inter-individual variation in normal bronchial epithelial cell (NBEC) cis-regulation of antioxidant, DNA repair, and cell cycle control genes are key determinants of lung cancer risk. Thus, there is a need for NBEC culture methods that enable extended population doublings without genetic alteration to enable experimental investigation of putative cis-regulatory SNPs in NBEC. Toward this goal, we assessed the effect of previously reported conditional reprogrammed culture (CRC) conditions on regulation of human telomerase reverse transcriptase (hTERT) transcript abundance and proliferative potential in NBEC. Methods. NBEC were obtained by bronchoscopic brush from eight individuals after obtaining informed consent to an IRB-approved protocol. NBEC were incubated in three different culture conditions: bronchial epithelial cell growth media (BEGM) only, co-cultured with irradiated mouse embryonic fibroblasts (IRR-MEF) + Rho kinase inhibitor (ROCKi) in BEGM, and conditioned BEGM + ROCKi. Media were changed every three days and cells were passaged and sub-cultured after ten days. Human telomerase reverse transcriptase (hTERT) was measured in three individuals after each passage in triplicate via qPCR. The proliferative capacity of all eight individuals was assessed using cell count and morphology at passage >3. Results. Co-culturing NBEC with IRR-MEF in BEGM supplemented with ROCKi produced a highly proliferative cell population while maintaining lineage commitment evident after removal of CRC conditions. Transcript abundance of hTERT was elevated 6.5-fold in NBEC in co-cultured conditions and 4.3-fold in NBEC in conditioned media compared to BEGM alone. Cell count in CRC conditions were up to 22-fold higher compared to BEGM alone. Cells were passaged and sub-cultured up to passage 4, followed by being frozen down in cell culture freezing media for further assessment. Conclusion. NBEC hTERT transcript abundance was up-regulated and cell population proliferative potential was extended in CRC conditions. It is likely that hTERT functions to protect the ends of linear chromosomes in dividing cells, enabling increased cell divisions while maintaining normal genome. These cell populations will be used in future studies to assess the effect of putative cis-regulatory single nucleotide polymorphisms (SNPs) on gene expression in NBEC. Citation Format: Daniel J. Craig, Rose T. Zolondek, Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, James C. Willey. Induction of hTERT and increased proliferative potential in conditionally reprogrammed normal bronchial epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2905.