Cigarette smoking is the primary cause of bronchogenic carcinoma (BC), yet only 10-15% of heavy s... more Cigarette smoking is the primary cause of bronchogenic carcinoma (BC), yet only 10-15% of heavy smokers develop BC and it is likely that this variation in risk is, in part, genetically determined. We previously reported a set of antioxidant genes for which transcript abundance was lower in normal bronchial epithelial cells (NBEC) of BC individuals compared to non-BC individuals. In unpublished studies of the same NBEC samples, transcript abundance values for several DNA repair genes were correlated with these antioxidant genes. From these data, we hypothesized that antioxidant and DNA repair genes are co-regulated by one or more transcription factors and that inter-individual variation in expression and/or function of one or more of these transcription factors is responsible for inter-individual variation in risk for BC. The putative transcription factor recognition sites common to six of the antioxidant genes were identified through in silico DNA sequence analysis. The transcript a...
A method that provides standardized data and is relatively inexpensive and capable of high throug... more A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.
Clinical implementation of Next-Generation Sequencing (NGS) is challenged by poor control for sto... more 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. 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. 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 (R(2) = 0.93). 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.
An outbreak of a unique and especially virulent strain (IVb) of viral hemorrhagic septicemia (VHS... more An outbreak of a unique and especially virulent strain (IVb) of viral hemorrhagic septicemia (VHS) occurred in 2005 in the Great Lakes, killing several economically prominent freshwater fishes; including yellow perch (Perca flavescens) and muskellunge (Esox masquinongy); with additional outbreaks in subsequent years. This virus poses extensive risk to the aquaculture, baitfish industries, and native fisheries. Despite efforts to reduce detection time with DNA diagnostics, cell culture, which is a weeks-long laborious process, is the only currently USDA-APHIS approved diagnostic method. Our laboratories have developed a new molecular based assay, Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR), to detect VHSv, which speeds detection time (to hours), lowers detection threshold, uniquely identifies among VHSv strains, determines whether the virus is actively replicating (transmissible), and provides intrinsic quality control via a standardized mixture of intern...
Cigarette smoking is the primary cause of bronchogenic carcinoma (BC), yet only 10-15% of heavy s... more Cigarette smoking is the primary cause of bronchogenic carcinoma (BC), yet only 10-15% of heavy smokers develop BC and it is likely that this variation in risk is, in part, genetically determined. We previously reported a set of antioxidant genes for which transcript abundance was lower in normal bronchial epithelial cells (NBEC) of BC individuals compared to non-BC individuals. In unpublished studies of the same NBEC samples, transcript abundance values for several DNA repair genes were correlated with these antioxidant genes. From these data, we hypothesized that antioxidant and DNA repair genes are co-regulated by one or more transcription factors and that inter-individual variation in expression and/or function of one or more of these transcription factors is responsible for inter-individual variation in risk for BC. The putative transcription factor recognition sites common to six of the antioxidant genes were identified through in silico DNA sequence analysis. The transcript a...
A method that provides standardized data and is relatively inexpensive and capable of high throug... more A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.
Clinical implementation of Next-Generation Sequencing (NGS) is challenged by poor control for sto... more 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. 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. 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 (R(2) = 0.93). 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.
An outbreak of a unique and especially virulent strain (IVb) of viral hemorrhagic septicemia (VHS... more An outbreak of a unique and especially virulent strain (IVb) of viral hemorrhagic septicemia (VHS) occurred in 2005 in the Great Lakes, killing several economically prominent freshwater fishes; including yellow perch (Perca flavescens) and muskellunge (Esox masquinongy); with additional outbreaks in subsequent years. This virus poses extensive risk to the aquaculture, baitfish industries, and native fisheries. Despite efforts to reduce detection time with DNA diagnostics, cell culture, which is a weeks-long laborious process, is the only currently USDA-APHIS approved diagnostic method. Our laboratories have developed a new molecular based assay, Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR), to detect VHSv, which speeds detection time (to hours), lowers detection threshold, uniquely identifies among VHSv strains, determines whether the virus is actively replicating (transmissible), and provides intrinsic quality control via a standardized mixture of intern...
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Papers by Erin Crawford