Xiaolu Zhang

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.

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.

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

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.

Abstract 2084: Genetic variation at a cis-acting C/EBPG binding site is associated with allele-specific ERCC5 transcript expression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: CCAAT/enhancer-binding protein gamma (C/EBPG) transcription factor expression is correlated with that of ERCC5 and other key DNA repair genes in normal bronchial epithelial cells (NBEC) suggesting a regulatory role. In prior studies, ERCC5 transcript expression was increased in a human lung carcinoma cell line H23 following CEBPG overexpression and in NBEC from 81 subjects, A allele at putative ERCC5 cis-regulatory SNP (rSNP) rs751402 and T allele at rSNP rs2296147 were associated with higher expression of ERCC5 marker SNP rs1047768 T allele transcript. rs751402 is located in open chromatin region identified by FAIRE-seq in NBEC and variation at rs751402 is predicted to alter binding of C/EBP. These studies support the hypothesis that allelic differential affinity to C/EBPG at rs751402 contributes to hereditary inter-individual variation in regulation of ERCC5 either directly or through interaction with complexes bound at rs2296147. The purpose of this study was to further investigate the role of C/EBPG in ERCC5 cis-regulation in an independent cohort of subjects and lung cancer cell lines. Methods: We knocked-down C/EBPG transcript level by C/EBPG siRNA transfection in human non-small cell lung carcinoma cell line H1703. Total and allele-specific expression (ASE) at rs1047768 was measured through multiplex competitive PCR-based amplicon sequencing library preparation followed by Illumina HiSeq next generation sequencing (NGS). This NGS 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. The genotype at rs751402 and rs2296147 in NBEC from 78 subjects and 14 human lung carcinoma cell lines was determined by TaqMan SNP genotyping assays. Direct assessment of the syntenic relationship of alleles in gDNA from poly-heterozygous individuals was assessed by allele-specific PCR followed by sequencing. Results: CEBPG transcript expression was knocked-down by 93% in H1703 cells and this was associated with 4-fold reduction in the ERCC5 transcript level at rs1047768. ERCC5 displayed significant inter-individual variation in allele specific expression (ASE) in NBEC from 85 subjects. Thirty nine out of 92 subjects including 3 cell lines were heterozygous at rs751402 and rs2296147 rSNPs and rs1047768 marker SNP and will be assessed for haplotypes comprising those sites. Conclusions: Results are consistent with CEBPG regulation of ERCC5 in the cell line H1703. The results obtained will enable us to test the hypothesis that haplotypes comprising particular alleles syntenic between rs1047768 and rs751402 are associated with higher allele-specific ERCC5 transcript abundance. Cell lines heterozygous at three sites will be subjected to CEBPG up and/or down regulation to assess effect on allele-specific ERCC5 expression at rs1047768. Citation Format: Xiaolu Zhang, Jiyoun Yeo, Erin Crawford, James C. Willey. Genetic variation at a cis-acting C/EBPG binding site is associated with allele-specific ERCC5 transcript expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2015-2084

Abstract 2205: Identification of transcript abundance difference at lung cancer and COPD risk genes in normal bronchial epithelial cells

Background: Lung cancer and chronic obstructive pulmonary disease (COPD) and are leading causes of morbidity and mortality both in the United States and worldwide. Inhalation of cigarette smoke is the primary known and preventable cause but only 10-15% of heavy smokers develop these diseases. This suggests that cigarette smoke exposure interacts with inherited susceptibility factors to determine risk. There is urgent need to identify heritable susceptibility factors that explain the majority of lung cancer and COPD risk. In prior studies, we and/or others have observed large inter-individual variation in normal bronchial epithelial cell (NBEC) expression of antioxidant, DNA repair, and cell regeneration control genes and altered regulation in NBEC of subjects with lung cancer or COPD. We collected normal bronchial epithelial cell (NBEC) samples from over 500 subjects with or demographically at risk for lung cancer. In this pilot study, we assessed total expression of multiple putative risk genes in NBEC samples from 78 subjects. Methods: A targeted competitive multiplex next generation sequencing (NGS) method (Blomquist et al, PLOS one 2013) was used to quantify transcript abundance at 70 marker sites among 33 target genes in NBEC total RNA from 78 subjects (18 cancer cases and 60 non-cancer controls). Approximately half of the cancer group (N = 10) and non-cancer group (N = 30) had COPD (FEV1/FVC Results: TP73 was expressed higher and XRCC1 and MUC5B lower in cancer. GSTP1, MUC5B, OGG1, and TP73 were expressed at significantly higher level in COPD vs control by t-test in non-cancer group. Conclusions: This study of 78 subjects provides evidence for transcript abundance difference in selected genes with high prior likelihood for contributing to lung cancer and COPD risk. Those genes will be the focus of additional studies in BEC samples from over 500 subjects with or at risk for lung cancer and/or COPD in effort to further optimize current tests for lung cancer and COPD risk. Note: This abstract was not presented at the meeting. Citation Format: Jiyoun Yeo, Xiaolu Zhang, Erin Crawford, James Willey. Identification of transcript abundance difference at lung cancer and COPD risk genes in normal bronchial epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2205. doi:10.1158/1538-7445.AM2015-2205

Abstract 3401: Inter-individual variation in MUC5B allele specific expression in normal bronchial epithelial cells and relationship to lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Familial interstitial pneumonia and idiopathic pulmonary fibrosis are associated with increased risk of lung cancer. Seibold et al (NEJM, 2011) recently identified a common variant in the putative promoter of MUC5B (rs35705950) associated with level of MUC5B expression in lung tissue and also with development of these diseases. The goal of this study was to determine whether the MUC5B rs35705950 variant is associated with a) MUC5B allele-specific expression (ASE) or total expression in normal bronchial epithelial cells (NBEC) or b) lung cancer risk. Through funding in part from RC2 CA148572 and HL108016 we collected NBEC samples from over 500 subjects with lung cancer or at risk for lung cancer. Methods: This was a pilot study of 85 subjects (26 cancer cases and 59 non-cancer controls). RNA was extracted from normal bronchial airway brush NBEC specimens of 85 subjects and reverse transcribed. Using next generation sequencing (NGS), allele-specific expression (ASE) was measured as allelic imbalance in each cDNA at three marker SNPs in MUC5B coding region (rs2075859, rs2943510, and rs4963059) selected for common (>0.05) minor allele frequency, using matched peripheral blood cell gDNA as a control. Specifically, each cDNA and matched gDNA sample was subjected to targeted competitive template multiplex PCR amplicon library generation followed by NGS (Blomquist et al, PLOS one, 2013) on Illumina Hiseq platform. This NGS method controlled 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. The genotype at putative cis-regulatory SNP rs35705950 was assessed in gDNA from 95 subjects (31 cancer cases and 64 non-cancer controls) including those assessed for ASE using a TaqMan® SNP assay. Results: There was significant (p 3 log) but not associated with lung cancer. Conclusions: These results support previous observations that there is significant inter-individual variation in cis-regulation of MUC5B in NBEC and suggest that this variation also may contribute to inherited lung cancer risk. Lack of association between rs35705950 genotype and MUC5B ASE may have been due to role of other cis-regulatory factors, and/or environmentally associated transfactors that contributed to large inter-individual variation in total MUC5B expression. Citation Format: Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, James C. Willey. Inter-individual variation in MUC5B allele specific expression in normal bronchial epithelial cells and relationship to lung cancer. [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 3401. doi:10.1158/1538-7445.AM2014-3401

Abstract 4251: Identification of expression quantitative trait loci at lung cancer and COPD risk genes in normal bronchial epithelial cells

Background: Lung cancer and chronic obstructive pulmonary disease (COPD) and are leading causes of morbidity and mortality both in the United States and worldwide. Inhalation of cigarette smoke is the primary known and preventable cause but only 10-15% of heavy smokers develop these diseases. This suggests that cigarette smoke exposure interacts with inherited susceptibility factors to determine risk. While some susceptibility genes are known, they account for less than 5% of risk for either disease. There is urgent need to identify heritable susceptibility factors that explain the majority of lung cancer and COPD risk. In this study we focused on discovery of cis-regulatory expression quantitative trait loci (eQTL) responsible for inter-individual variation in the expression of genes that we and/or others have reported to display altered regulation in subjects with lung cancer or COPD. Through funding in part from RC2 CA148572 and HL108016 we collected normal bronchial epithelial cell (NBEC) samples from over 500 subjects with lung cancer or COPD or demographically at risk for lung cancer or COPD. In this pilot study, we assessed allele-specific expression (ASE) and total expression of multiple putative risk genes in NBEC samples from 85 subjects. Methods: A targeted competitive multiplex next generation sequencing (NGS) method (STAndardized RNA SEQuencing [STARSEQ]; Blomquist et al, PLOS one 2013) was used to quantify a) allele specific expression (ASE) and total expression at 140 single nucleotide polymorphism (SNP) sites among 41 target genes (including eleven of 14 genes comprised by the previously reported lung cancer risk test (Blomquist et al, Can. Res. 2009) in NBEC total RNA from 85 subjects (26 cancer cases and 59 non-cancer controls), and b) allelic representation and putative cis-regulatory single nucleotide polymorphisms (rSNPs) in matched peripheral blood leukocyte genomic (g)DNA from the same subjects. Heterozygosity was determined by gDNA analysis. Results: Significant (p Conclusions: This small study of 85 subjects provides evidence for cis-regulatory eQTL in selected genes with high prior likelihood for contributing to lung cancer and COPD risk. The genes with ASE reported here will be among those included in additional studies in archived NBEC samples from 500 subjects aiming to further optimize current tests for lung cancer and COPD risk and better understand mechanisms of risk. Citation Format: Erin L. Crawford, Jiyoun Yeo, Xiaolu Zhang, Karan Padda, Taylor Arend, Thomas M. Blomquist, Albert M. Levin, Mei Lu, James C. Willey. Identification of expression quantitative trait loci at lung cancer and COPD risk genes in normal bronchial epithelial 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 4251. doi:10.1158/1538-7445.AM2014-4251