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Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing

Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈106 cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100× were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100× were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations

Data-Retention-Voltage-Based Analysis of Systematic Variations in SRAM SEU Hardness: A Possible Solution to Synergistic Effects of TID

Single-event upset (SEU) hardness varies across dies, wafers, and lots—even just after fabrication and further across time. Mechanisms of postfabrication variations include total ionizing dose (TID) effects, which are caused by long-term radiation exposure. This synergistic effect of TID on SEU hardness is a particular concern in integrated circuits used in space and nuclear radiation environments. This article shows that an electrical parameter called the data-retention voltage is useful in dealing with such TID effects on the SEU hardness of static random access memories (SRAMs), which are known to be particularly radiation-sensitive. Experiments showed that TID-induced variations in SRAM SEU hardness, i.e., variations in SEU cross sections, were predicted by measuring the data-retention voltage. In addition, these variations were canceled out by adjusting the power supply voltage according to its interesting relationship to the data-retention voltage. Results suggest that it might be possible in flight to predict and cancel out SEU hardness variations caused by TID and other synergistic effects

Investigation of Buried-Well Potential Perturbation Effects on SEU in SOI DICE-Based Flip-Flop Under Proton Irradiation

:The effects of buried-well potential perturbation under the buried-oxide (BOX) layer are studied in both a heavy-ion single event upset (SEU) test and a high-energy proton-SEU test of a silicon-on-insulator (SOI) dual interlocked storage cell (DICE)-based flip-flop. Their dependence on incident angle and back bias is discussed. We fabricated both DICE-based flip-flop and conventional flip-flop, which are designed as 80 000-stage shift-register chains. In a heavy-ion test, a considerable number of SEUs were observed at back bias exceeding 2.4 V, and a ten-times larger SEU-cross section was finally recorded at back bias of 3.0 V compared with the total active area of a DICE-based flip-flop cell. This marks the first case where DICE topology was found to be broken by buried-well potential perturbation on an SOI DICE-based flip-flop. In a proton test, one error was observed at back bias of 2.0 V. The SEU rate in the Van Allen belt at an altitude of 2300 km and an inclination of 90° was estimated as being once every 5 years