Advancing NGS quality control to enable measurement of actionable mutations in circulating tumor DNA.

The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next-generation sequencing (NGS) method that will enable more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. To accomplish this, a synthetic internal standard spike-in was designed for each actionable mutation target, suitable for use in NGS following hybrid capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position in each sample. True-positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

New Tool for Monitoring Molecular Response in Patients With Chronic Myeloid Leukemia.

OBJECTIVE: Chronic myeloid leukemia treatment monitoring using polymerase chain reaction-based peripheral blood testing of t9;22 BCR-ABL1 provides improved test sensitivity over cytology but suffers from inadequate standardization in most laboratories due to variations inherent in the existing polymerase chain reaction methodologies. We performed the initial analytic performance evaluation of a novel competitive template-based peripheral blood b2a2/b3a2 transcript abundance method, called standardized nucleic acid quantification (SNAQ) test, with hypothesis that this will produced more consistent results with less frequent interlaboratory variations. MATERIALS AND METHODS: Thirty-six chronic myeloid leukemia patients treated at our institution were enrolled. We compared SNAQ test with 2 laboratory developed test at the MD Anderson molecular diagnostic laboratory and Cancer Genetics Institute for analyzing BCR-ABL1 from peripheral blood samples. Each test result (n=36) was ranked against all the other samples tested by the same method. RESULTS: The Pearson correlation between SNAQ and laboratory developed test done at 2 labs was met by correlations of 0.97, 0.96, 0.96, and 0.94. Analysis of variance of log %BCR-ABL1 interlaboratory results indicated no significant difference (P=0.98). Post hoc analysis of method agreement showed the SNAQ method had a 95% limit of agreement of ±3-fold between laboratories. CONCLUSIONS: In this pilot study, SNAQ methodology performed consistent with half-log accuracy. Additional studies from a larger sample size and correlation with clinical outcomes are required to confirm this observation.

Proof of concept study to assess fetal gene expression in amniotic fluid by nanoarray PCR.

Microarray analysis of cell-free RNA in amniotic fluid (AF) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype. Once informative genes are identified, research moves to a more focused platform such as quantitative reverse transcriptase-PCR. Standardized NanoArray PCR (SNAP) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids. We used a previously developed SNAP gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant. RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation, and transcript abundance of 21 genes was measured. Statistically significant differences in expression, as a function of advancing gestational age, were observed for 5 of 21 genes. ANXA5, GUSB, and PPIA showed decreasing gene expression over time, whereas CASC3 and ZNF264 showed increasing gene expression over time. Statistically significantly increased expression of MTOR and STAT2 was seen in female compared with male fetuses. This study demonstrates the feasibility of focused fetal gene expression analysis using SNAP technology. In the future, this technique could be optimized to examine specific genes instrumental in fetal organ system function, which could be a useful addition to prenatal care.

Nanoliter high-throughput RT-qPCR: a statistical analysis and assessment.

Biomarkers discovered from gene expression profiles using hybridization microarrays have made great inroads in the diagnosis and development of safer and efficacious drugs. The candidate gene set is biologically validated by quantitative measurement with reverse transcriptase quantitative PCR (RT-qPCR) and is an effective strategy when implemented with microplates if the number of candidate genes and samples is small. With the trend toward informative candidate gene panels increasing from tens to hundreds of genes and sample cohorts exceeding several hundred, an alternative fluidic approach is needed that preserves the intrinsic analytical precision, large dynamic range, and high sensitivity of RT-qPCR, yet is scalable to high throughputs. We have evaluated the performance of a nanoliter fluidic system that enables up to 3072 nanoliter RT-qPCR assays simultaneously in a high-density array format. We measured the transcription from two different adult human tissues to assess measurement reproducibility across replicates, measurement accuracy, precision, specificity, and sensitivity; determined the false positive rate (FPR) and false negative rate (FNR) of the expressed transcript copies; and determined differences in kinase gene expression reflecting tissue and dosage differences. Using our methodology, we confirm the potential of this technology in advancing pharmaceutical research and development.

A nanoliter fluidic platform for large-scale single nucleotide polymorphism genotyping.

Discovery, evaluation, and understanding the biological relevance of single nucleotide polymorphisms (SNPs) and their associated phenotypes is relevant to many applications, including human disease diagnostics, pathogen detection, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency. Validation of putative SNP associations in large-scale cohorts is currently impeded by the technical challenges and high cost inherent in analyzing large numbers of samples using available SNP genotyping platforms. We describe in this report the implementation of the 5'-exonuclease, biallelic PCR assay for SNP genotyping (TaqMan) in a nanofluidic version of a high-density microplate. System performance was assessed using a panel of 32 TaqMan SNP genotyping assays targeted to human polymorphisms. This functional test of the nanoliter fluidic SNP genotyping platform delivered genotyping call rates and accuracies comparable to the same larger volume reactions in microplate systems.

Chemotactic signaling by an Escherichia coli CheA mutant that lacks the binding domain for phosphoacceptor partners.

CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheADeltaP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheADeltaP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheADeltaP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheADeltaP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.