Molecular sampling at logarithmic rates for next-generation sequencing.

Next-generation sequencing is a cutting edge technology, but to quantify a dynamic range of abundances for different RNA or DNA species requires increasing sampling depth to levels that can be prohibitively expensive due to physical limits on molecular throughput of sequencers. To overcome this problem, we introduce a new general sampling theory which uses biophysical principles to functionally encode the abundance of a species before sampling, SeQUential depletIon and enriCHment (SQUICH). In theory and simulation, SQUICH enables sampling at a logarithmic rate to achieve the same precision as attained with conventional sequencing. A simple proof of principle experimental implementation of SQUICH in a controlled complex system of ~262,000 oligonucleotides already reduces sequencing depth by a factor of 10. SQUICH lays the groundwork for a general solution to a fundamental problem in molecular sampling and enables a new generation of efficient, precise molecular measurement at logarithmic or better sampling depth.

Hepatitis C virus direct-acting antiviral nonadherence: Relationship to sustained virologic response and identification of at-risk patients.

OBJECTIVES: There is a dearth of literature on effects of nonadherence to hepatitis C virus (HCV) direct-acting antiviral (DAA) regimens; thus, the objective of our study was to assess the impact of adherence on sustained virologic response (SVR) and evaluate factors associated with nonadherence, such as race, psychiatric comorbidities, and therapy length. METHODS: We conducted a retrospective cohort study of patients completing DAA treatment between January 2014 and May 2016 within an interdisciplinary hepatology clinic. Adherence was defined a priori as 95% or greater of DAA doses taken within the prescribed treatment period. Post hoc analyses were done with adherence thresholds ≥ 90%, ≥ 85%, and ≥ 80% and adherence as a continuous percentage. Patients lost to follow-up before completing therapy or that discontinued therapy early were excluded from analyses. The association between adherence and SVR rates was assessed using Fisher exact test (for adherence thresholds) and the Wilcoxon rank-sum test (for continuous adherence). Factors associated with adherence were assessed similarly using Fisher exact and Wilcoxon rank-sum tests and multivariable logistic regression. RESULTS: Overall adherence was high, with an average of 97.8% of DAA doses taken within the prescribed treatment period. Achievement of SVR was not significantly different in adherent and nonadherent patients, at an adherence threshold of 95% or greater (93.4% vs. 88.5%; P = 0.246) or any of the post hoc adherence thresholds (≥ 90% [93.3% vs. 84.0%; P = 0.098], ≥ 85% [92.8% vs. 91.7%; P = 0.601], ≥ 80% [92.9% vs. 80.0%; P = 0.315], or as a continuous percentage [P = 0.328]). Black patients were significantly more likely to be nonadherent to DAAs than non-black patients at each adherence threshold (P < 0.05). No other factors evaluated were associated with nonadherence. CONCLUSION: A numerically higher but not statistically significant SVR failure rate was noted in nonadherent patients, although the gold standard definition for adherence remains to be established. Black patients may require additional adherence support.

ciRS-7 exonic sequence is embedded in a long non-coding RNA locus.

ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. To study the biogenesis of ciRS-7, we developed an algorithm to define its promoter and predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generates a new model for regulation of this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChIP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.

Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.

With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.