Next-generation sequencing and its clinical application

Pyrosequencing is a new technology and can be used for mutation tests. However, its data analysis is a manual process and involves sophisticated algorithms. During this process, human errors may occur. A better way of analyzing pyrosequencing data is needed in clinical diagnostic laboratory. Computer software is potentially useful for pyrosequencing data analysis. We have developed such software, which is able to perform pyrosequencing mutation data analysis for epidermal growth factor receptor, Kirsten rat sarcoma viral oncogene homolog and v-raf murine sarcoma viral oncogene homolog B1. The input data for analysis includes the targeted nucleotide sequence, common mutations in the targeted sequence, pyrosequencing dispensing order, pyrogram peak order and peak heights. The output includes mutation type and percentage of mutant gene in the specimen.

Background: The detection of CEBPA and FLT3 mutations by next generation sequencing (NGS) is challenging due to high GC content and Internal Tandem Duplications (ITDs). Recent advances have been made to surmount these challenges. In this study, we compare three commercial kits and evaluate the performance of these more advanced hybrid-capture and AMP-chemistry based methods. Methods: Amplicon-based TSM 54-Gene Panel (Illumina) was evaluated against hybridization-capture SOPHiA Genetics MSP, OGT SureSeq, and AMP chemistry-based VariantPlex (Archer) for wet-lab workflow and data-analysis pipelines. Standard kit directions and commercial analysis pipelines were followed. Seven CEBPA and 10 FLT3-positive cases were identified that previously were missed on an amplicon NGS assay. The average reads, coverage uniformity, and the detection of CEBPA or FLT3 mutations were compared. Results: All three panels detected all 10 CEBPA mutations and all 10 FLT3 ITDs with 100% sensitivity. In addition, there was high concordance (100%) between all three panels detecting 47/47 confirmed variants in a set of core myeloid genes. Conclusions: The results show that the NGS assays are now able to reliably detect CEBPA mutations and FLT3 ITDs. These assays may allow foregoing additional orthogonal testing for CEBPA and FLT3.

One of the major quality assurance (QA) goals in many molecular laboratories is to avoid sample pipetting errors on the lab bench; especially when pipetting into multiwell plates. A pipetting error can cause a switch in patient samples, which can lead to recording the wrong results for the patient samples involved. Such pipetting errors are difficult to identify when it happens in lab bench work. DNA fingerprinting is a powerful tool in determining sample identities. Our laboratory has explored the usage of this technology in our QA process and successfully established that DNA fingerprinting can be used to monitor possible sample switch in gene rearrangement lab bench work. We use florescent light to quench the florescence in the gene rearrangement polymerase chain reaction products. After that, DNA fingerprinting technology is used to identify the sample DNA in the gene rearrangement polymerase chain reaction plate. The result is compared with the corresponding patient’s blood sample DNA to determine whether there is a sample switch during the lab bench work.

The radiation sensitivity index (RSI) and 12-chemokine gene expression signature (12CK GES) are two gene expression signatures (GES) that were previously developed to predict tumor radiation sensitivity or identify the presence of tertiary lymphoid structures in tumors, respectively. To advance the use of these GESs into clinical trial evaluation, their assays must be assessed within the context of the Clinical Laboratory Improvement Amendments (CLIA) process. Using HG-U133Plus2.0 arrays, we first established CLIA laboratory proficiency. Then the accuracy (limit of detection and macrodissection impact), precision (variability by time and operator), sample type (surgery vs. biopsy), and concordance with a reference laboratory were evaluated. RSI and 12CK GES were reproducible (RSI: 0.01 mean difference, 12CK GES: 0.17 mean difference) and precise with respect to time and operator. Taken together, the reproducibility analysis of the scores indicated a median RSI difference of 0.06 (6.47% of range) across samples and a median 12CK GES difference of 0.92 (12.29% of range). Experiments indicated that the lower limit of input RNA is 5 ng. Reproducibility with a second CLIA laboratory demonstrated reliability with the median RSI score difference of 0.065 (6% of full range) and 12CK GES difference of 0.93 (12% of observed range). Overall, under CLIA, RSI and 12CK GES were demonstrated by the Moffitt Cancer Center Advanced Diagnostic Laboratory to be reproducible GESs for clinical usage. Significance: The RSI and 12CK GES are two GESs that predict tumor radiation sensitivity or the presence of tertiary lymphoid structures in tumors, respectively. These GESs were assessed within the CLIA process for future clinical use. We established proficiency, reproducibility, and reliability characteristics for both signatures in a controlled setting, indicating these GESs are suitable for validation within future clinical trials.