Clinical validation of the tempus xT next-generation targeted oncology sequencing assay.

We developed and clinically validated a hybrid capture next generation sequencing assay to detect somatic alterations and microsatellite instability in solid tumors and hematologic malignancies. This targeted oncology assay utilizes tumor-normal matched samples for highly accurate somatic alteration calling and whole transcriptome RNA sequencing for unbiased identification of gene fusion events. The assay was validated with a combination of clinical specimens and cell lines, and recorded a sensitivity of 99.1% for single nucleotide variants, 98.1% for indels, 99.9% for gene rearrangements, 98.4% for copy number variations, and 99.9% for microsatellite instability detection. This assay presents a wide array of data for clinical management and clinical trial enrollment while conserving limited tissue.

Quantitative polymerase chain reaction using the comparative C q method.

Quantitative PCR (qPCR) is considered the gold standard for molecular DNA quantification and can be used for a wide range of techniques from comparing gene expression levels to quantifying DNA copy number variation. The strengths of this assay include sensitivity to a wide range of expression levels, low starting template requirement, which is important when samples are scarce, and quick turnaround time. However, there are many variables to consider when performing qPCR: including (1) starting materials (e.g., tissues, cells, or genomic DNA), (2) fluorescent detection (e.g., SYBR green dye, fluorescent probes, or multiplexed assays), and (3) analysis methods (e.g., simple equations with single reference genes or complex algorithms with multiple reference genes). This chapter will introduce the process of designing an experiment while avoiding common mistakes and present tools for performing qPCR in a practical, simple, and efficient manner.

Identification of PVT1 as a candidate gene for end-stage renal disease in type 2 diabetes using a pooling-based genome-wide single nucleotide polymorphism association study.

To identify genetic variants contributing to end-stage renal disease (ESRD) in type 2 diabetes, we performed a genome-wide analysis of 115,352 single nucleotide polymorphisms (SNPs) in pools of 105 unrelated case subjects with ESRD and 102 unrelated control subjects who have had type 2 diabetes for > or =10 years without macroalbuminuria. Using a sliding window statistic of ranked SNPs, we identified a 200-kb region on 8q24 harboring three SNPs showing substantial differences in allelic frequency between case and control pools. These SNPs were genotyped in individuals comprising each pool, and strong evidence for association was found with rs2720709 (P = 0.000021; odds ratio 2.57 [95% CI 1.66-3.96]), which is located in the plasmacytoma variant translocation gene PVT1. We sequenced all exons, exon-intron boundaries, and the promoter of PVT1 and identified 47 variants, 11 of which represented nonredundant markers with minor allele frequency > or =0.05. We subsequently genotyped these 11 variants and an additional 87 SNPs identified through public databases in 319-kb flanking rs2720709 ( approximately 1 SNP/3.5 kb); 23 markers were associated with ESRD at P < 0.01. The strongest evidence for association was found for rs2648875 (P = 0.0000018; 2.97 [1.90-4.65]), which maps to intron 8 of PVT1. Together, these results suggest that PVT1 may contribute to ESRD susceptibility in diabetes.