Clinical applications of precision oncology require accurate tests that can distinguish true cancer specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

BackgroundToll‐like receptors (TLRs) have been shown to be involved in cerebral ischemia/reperfusion (I/R) injury. TLR9 is located in intracellular compartments and recognizes CpG‐DNA. This study examined the effect of CpG‐ODN on cerebral I/R injury.Methods and ResultsC57BL/6 mice were treated with CpG‐ODN by i.p. injection 1 hour before the mice were subjected to cerebral ischemia (60 minutes) followed by reperfusion (24 hours). Scrambled‐ODN served as control‐ODN. Untreated mice, subjected to cerebral I/R, served as I/R control. The effect of inhibitory CpG‐ODN (iCpG‐ODN) on cerebral I/R injury was also examined. In addition, we examined the therapeutic effect of CpG‐ODN on cerebral I/R injury by administration of CpG‐ODN 15 minutes after cerebral ischemia. CpG‐ODN administration significantly decreased cerebral I/R‐induced infarct volume by 69.7% (6.4±1.80% vs 21.0±2.85%, P<0.05), improved neurological scores, and increased survival rate, when compared with the untreated I/R group. Therapeutic administration of CpG‐ODN also significantly reduced infarct volume by 44.7% (12.6±2.03% vs 22.8±2.54%, P<0.05) compared with untreated I/R mice. Neither control‐ODN, nor iCpG‐ODN altered I/R‐induced cerebral injury or neurological deficits. Nissl staining showed that CpG‐ODN treatment preserved neuronal morphology in the ischemic hippocampus. Immunoblot showed that CpG‐ODN administration increased Bcl‐2 levels by 41% and attenuated I/R‐increased levels of Bax and caspase‐3 activity in ischemic brain tissues. Importantly, CpG‐ODN treatment induced Akt and GSK‐3β phosphorylation in brain tissue and cultured microglial cells. PI3K inhibition with LY294002 abolished CpG‐ODN‐induced protection.ConclusionCpG‐ODN significantly reduces cerebral I/R injury via a PI3K/Akt‐dependent mechanism. Our data also indicate that CpG‐ODN may be useful in the therapy of cerebral I/R injury.

African Americans carrying two apolipoprotein L1 gene (APOL1) renal risk variants have a high risk for nephropathy. However, only a minority develops end-stage renal disease (ESRD). Hence, modifying factors likely contribute to initiation of kidney disease such as endogenous (HIV infection) or exogenous (interferon treatment) environmental modifiers. In this report, genome-wide association studies and a meta-analysis were performed to identify novel loci for nondiabetic ESRD in African Americans and to detect genetic modifiers in APOL1-associated nephropathy. Two African American cohorts were analyzed, 1749 nondiabetic ESRD cases and 1136 controls from Wake Forest and 901 lupus nephritis (LN)-ESRD cases and 520 controls with systemic lupus erythematosus but lacking nephropathy from the LN-ESRD Consortium. Association analyses adjusting for APOL1 G1/G2 renal-risk variants were completed and stratified by APOL1 risk genotype status. Individual genome-wide association studies and meta-analysis results of all 2650 ESRD cases and 1656 controls did not detect significant genome-wide associations with ESRD beyond APOL1. Similarly, no single nucleotide polymorphism showed significant genome-wide evidence of an interaction with APOL1 risk variants. Thus, although variants with small individual effects cannot be ruled out and are likely to exist, our results suggest that APOL1-environment interactions may be of greater clinical importance in triggering nephropathy in African Americans than APOL1 interactions with other single nucleotide polymorphisms.

Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.