ClonEvol: clonal ordering and visualization in cancer sequencing.

BACKGROUND: Reconstruction of clonal evolution is critical for understanding tumor progression and implementing personalized therapies. This is often done by clustering somatic variants based on their cellular prevalence estimated via bulk tumor sequencing of multiple samples. The clusters, consisting of the clonal marker variants, are then ordered based on their estimated cellular prevalence to reconstruct clonal evolution trees, a process referred to as 'clonal ordering'. However, cellular prevalence estimate is confounded by statistical variability and errors in sequencing/data analysis, and therefore inhibits accurate reconstruction of the clonal evolution. This problem is further complicated by intra- and inter-tumor heterogeneity. Furthermore, the field lacks a comprehensive visualization tool to facilitate the interpretation of complex clonal relationships. To address these challenges we developed ClonEvol, a unified software tool for clonal ordering, visualization, and interpretation. MATERIALS AND METHODS: ClonEvol uses a bootstrap resampling technique to estimate the cellular fraction of the clones and probabilistically models the clonal ordering constraints to account for statistical variability. The bootstrapping allows identification of the sample founding- and sub-clones, thus enabling interpretation of clonal seeding. ClonEvol automates the generation of multiple widely used visualizations for reconstructing and interpreting clonal evolution. RESULTS: ClonEvol outperformed three of the state of the art tools (LICHeE, Canopy and PhyloWGS) for clonal evolution inference, showing more robust error tolerance and producing more accurate trees in a simulation. Building upon multiple recent publications that utilized ClonEvol to study metastasis and drug resistance in solid cancers, here we show that ClonEvol rediscovered relapsed subclones in two published acute myeloid leukemia patients. Furthermore, we demonstrated that through noninvasive monitoring ClonEvol recapitulated the emerging subclones throughout metastatic progression observed in the tumors of a published breast cancer patient. CONCLUSIONS: ClonEvol has broad applicability for longitudinal monitoring of clonal populations in tumor biopsies, or noninvasively, to guide precision medicine. AVAILABILITY: ClonEvol is written in R and is available at https://github.com/ChrisMaherLab/ClonEvol.

Rare, low frequency and common coding variants in CHRNA5 and their contribution to nicotine dependence in European and African Americans.

The common nonsynonymous variant rs16969968 in the α5 nicotinic receptor subunit gene (CHRNA5) is the strongest genetic risk factor for nicotine dependence in European Americans and contributes to risk in African Americans. To comprehensively examine whether other CHRNA5 coding variation influences nicotine dependence risk, we performed targeted sequencing on 1582 nicotine-dependent cases (Fagerström Test for Nicotine Dependence score⩾4) and 1238 non-dependent controls, with independent replication of common and low frequency variants using 12 studies with exome chip data. Nicotine dependence was examined using logistic regression with individual common variants (minor allele frequency (MAF)⩾0.05), aggregate low frequency variants (0.05>MAF⩾0.005) and aggregate rare variants (MAF<0.005). Meta-analysis of primary results was performed with replication studies containing 12 174 heavy and 11 290 light smokers. Next-generation sequencing with 180 × coverage identified 24 nonsynonymous variants and 2 frameshift deletions in CHRNA5, including 9 novel variants in the 2820 subjects. Meta-analysis confirmed the risk effect of the only common variant (rs16969968, European ancestry: odds ratio (OR)=1.3, P=3.5 × 10(-11); African ancestry: OR=1.3, P=0.01) and demonstrated that three low frequency variants contributed an independent risk (aggregate term, European ancestry: OR=1.3, P=0.005; African ancestry: OR=1.4, P=0.0006). The remaining 22 rare coding variants were associated with increased risk of nicotine dependence in the European American primary sample (OR=12.9, P=0.01) and in the same risk direction in African Americans (OR=1.5, P=0.37). Our results indicate that common, low frequency and rare CHRNA5 coding variants are independently associated with nicotine dependence risk. These newly identified variants likely influence the risk for smoking-related diseases such as lung cancer.

Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila--part I.

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.