We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.

The maintenance of intestinal homeostasis depends on a complex interaction between microbiota, intestinal epithelial barrier, and the immune system. Alteration in of one of these components could lead to the development of inflammatory bowel diseases (IBD). Norovirus infection of mice with a mutation in the Crohn’s disease susceptibility gene Atg16L1 induces intestinal inflammation. Moreover, persistent norovirus infection leads to intestinal virus-specific CD8+ T cells responses. However, the role of the enteric virome in IBD is still poorly understood. Toll-like receptor 7 (TLR7) recognizes single-stranded RNA viruses. Here, we investigate the role of TLR7 in intestinal homeostasis and inflammation. At steady state, Tlr7−/− mice have an approx. 10-fold increase in small and large intestinal lamina propria (LP) granzyme B+ tissue-resident memory (Trm) CD8+ T cells compared to WT mice (WT: 5.5, Tlr7−/−: 59.5 %, p < 0.005), reminiscent of persistent norovirus infection. Furthermore, Tlr7−/− mice were more susceptible to dextran sulfate sodium (DSS) colitis with more severe inflammation (Histoscore: WT: 7.6, Tlr7−/−: 12.7, p < 0.005), increased disease activity index (WT: 5.5, Tlr7−/−: 7.4, p < 0.05), and increased secretion of IFNg (WT: 5.2, Tlr7−/−: 24.2 ng/ml, p < 0.005) and TNFα (WT: 108.6, Tlr7−/−: 191.8 pg/ml, p < 0.05). Increased colonic inflammation was associated with increased LP Trm CD8+ T cells (WT: 3.9, Tlr7−/−: 42.0 %, p < 0.005). Our data shows that TLR7-deficiency promotes the development of LP Trm CD8+ T cells and increases susceptibility to DSS colitis. In conclusion, TLR7 plays an important role in maintaining immune response to intestinal viruses and protects against development of colitis.

No abstract