Targeted deep sequencing from multiple sources demonstrates increased NOTCH1 alterations in lung cancer patient plasma.

INTRODUCTION: Targeted therapies are based on specific gene alterations. Various specimen types have been used to determine gene alterations, however, no systemic comparisons have yet been made. Herein, we assessed alterations in selected cancer-associated genes across varying sample sites in lung cancer patients. MATERIALS AND METHODS: Targeted deep sequencing for 48 tumor-related genes was applied to 153 samples from 55 lung cancer patients obtained from six sources: Formalin-fixed paraffin-embedded (FFPE) tumor tissues, pleural effusion supernatant (PES) and pleural effusion cell sediments (PEC), white blood cells (WBCs), oral epithelial cells (OECs), and plasma. RESULTS: Mutations were detected in 96% (53/55) of the patients and in 83% (40/48) of the selected genes. Each sample type exhibited a characteristic mutational pattern. As anticipated, TP53 was the most affected sequence (54.5% patients), however this was followed by NOTCH1 (36%, across all sample types). EGFR was altered in patient samples at a frequency of 32.7% and KRAS 10.9%. This high EGFR/ low KRAS frequency is in accordance with other TCGA cohorts of Asian origin but differs from the Caucasian population where KRAS is the more dominant mutation. Additionally, 66% (31/47) of PEC samples had copy number variants (CNVs) in at least one gene. Unlike the concurrent loss and gain in most genes, herein NOTCH1 loss was identified in 21% patients, with no gain observed. Based on the relative prevalence of mutations and CNVs, we divided lung cancer patients into SNV-dominated, CNV-dominated, and codominated groups. CONCLUSIONS: Our results confirm previous reports that EGFR mutations are more prevalent than KRAS in Chinese lung cancer patients. NOTCH1 gene alterations are more common than previously reported and reveals a role of NOTCH1 modifications in tumor metastasis. Furthermore, genetic material from malignant pleural effusion cell sediments may be a noninvasive manner to identify CNV and participate in treatment decisions.

Spatial Vulnerabilities of the Escherichia coli Genome to Spontaneous Mutations Revealed with Improved Duplex Sequencing.

Investigation of spontaneous mutations by next-generation sequencing technology has attracted extensive attention lately due to the fundamental roles of spontaneous mutations in evolution and pathological processes. However, these studies only focused on the mutations accumulated through many generations during long-term (possibly be years of) culturing, but not the freshly generated mutations that occur at very low frequencies. In this study, we established a molecularly barcoded deep sequencing strategy to detect low abundant spontaneous mutations in genomes of bacteria cell cultures. Genome-wide spontaneous mutations in 15 Escherichia coli cell culture samples were defined with a high confidence (P < 0.01). We also developed a hotspot-calling approach based on the run-length encoding algorithm to find the genomic regions that are vulnerable to the spontaneous mutations. The hotspots for the mutations appeared to be highly conserved across the bacteria samples. Further biological annotation of these regions indicated that most of the spontaneous mutations were located at the repeat domains or nonfunctional domains of the genomes, suggesting the existence of mechanisms that could somehow prevent the occurrence of mutations in crucial genic areas. This study provides a more faithful picture of mutation occurrence and spectra in a single expansion process without long-term culturing.