Surveys of transcription in many organisms have observed widespread expression of RNAs with no known function, encoded within and between canonical coding genes. The search to distinguish functional RNAs from transcriptional noise represents one of the great challenges in genomic biology. Here we report a next-generation sequencing technique designed to facilitate the inference of function of uncharacterized transcript forms by improving their coverage in sequencing libraries, in parallel with the detection of canonical mRNAs. We piloted this protocol, which is based on the capture of 39 ends of polyadenylated RNAs, in budding yeast. Analysis of transcript ends in coding regions uncovered hundreds of alternative-length coding forms, which harbored a unique sequence motif and showed signatures of regulatory function in particular gene categories; independent single-gene measurements confirmed the differential regulation of short coding forms during heat shock. In addition, our 39-end RNA-seq method applied to wild-type strains detected putative noncoding transcripts previously reported only in RNA surveillance mutants, and many such transcripts showed differential expression in yeast cultures grown under chemical stress. Our results underscore the power of the 39-end protocol to improve detection of noncanonical transcript forms in a sequencing experiment of standard depth, and our findings strongly suggest that many unannotated, polyadenylated RNAs may have as yet uncharacterized regulatory functions.

Gene expression varies widely between individuals of a population, and regulatory change can underlie phenotypes of evolutionary and biomedical relevance. A key question in the field is how DNA sequence variants impact gene expression, with most mechanistic studies to date focused on the effects of genetic change on regulatory regions upstream of protein-coding sequence. By contrast, the role of RNA 3′-end processing in regulatory variation remains largely unknown, owing in part to the challenge of identifying functional elements in 3′ untranslated regions. In this work, we conducted a genomic survey of transcript ends in lymphoblastoid cells from genetically distinct human individuals. Our analysis mapped the cis-regulatory architecture of 3′ gene ends, finding that transcript end positions did not fall randomly in untranslated regions, but rather preferentially flanked the locations of 3′ regulatory elements, including miRNA sites. The usage of these transcript length forms and motifs varied across human individuals, and polymorphisms in polyadenylation signals and other 3′ motifs were significant predictors of expression levels of the genes in which they lay. Independent single-gene experiments confirmed the effects of polyadenylation variants on steady-state expression of their respective genes, and validated the regulatory function of 3′ cis-regulatory sequence elements that mediated expression of these distinct RNA length forms. Focusing on the immune regulator IRF5, we established the effect of natural variation in RNA 3′-end processing on regulatory response to antigen stimulation. Our results underscore the importance of two mechanisms at play in the genetics of 3′-end variation: the usage of distinct 3′-end processing signals and the effects of 3′ sequence elements that determine transcript fate. Our findings suggest that the strategy of integrating observed 3′-end positions with inferred 3′ regulatory motifs will prove to be a critical tool in continued efforts to interpret human genome variation.

Hadamard transform (HT) time-of-flight mass spectrometry (TOFMS) is a multiplexing technique that offers high duty cycle for the mass analysis of continuous ion sources. The multiplexing advantage is maximized when spectral noise is independent of signal intensity. For conditions in which shot noise predominates, the variance in each peak is a function of the population of all measured species. We develop expressions for the performance of a HT-TOF mass spectrometer based on Poissonian statistics for the arrival times of ions at the detector. These expressions and complementary probabilistic simulations are used to estimate the magnitude of the baseline noise as a function of mass spectral features and acquisition conditions. Experiment validates the predictions that noise depends on the total number of ions in the acquired spectrum, and the achieved signal-to-noise ratio for a given species depends on its relative population. We find that for HT-TOFMS experiments encoded with an n-order binary off-on sequence that contains N ϭ 2 n Ϫ 1 elements, the peak height precision, which is the peak intensity divided by its standard deviation, is greater than that of an equivalent conventional TOF experiment by a factor of ͙ N⁄2 times the square root of the fractional abundance of the peak of interest. Thus, HT-TOFMS is superior to conventional TOF for all species whose fractional abundance F i exceeds 2/N, which for a typical N value of 2047 corresponds to [1,2] is based on the pseudorandom gating of ion packets into a time-of-flight analyzer. In its typical implementation, the technique is able to monitor continuous ion sources with a 50% duty cycle, by which we mean the sample utilization efficiency, independent of all other figures of merit. Recently, we have demonstrated that the duty cycle can be extended to 100% using patterned, twochannel detection [3]. While a conventional TOFMS experiment releases a single packet of ions and makes independent measurements of all species, an HT-TOFMS experiment releases thousands of packets and measures the intensities of species as linear sums. For a fixed acquisition time, this procedure leads to linear increases in signal intensities and a root mean square reduction in the relative magnitude of signal-independent noise. Signal-dependent noise is known to affect the achieved performance of HT-TOFMS [4], but these processes have not been carefully studied.The principles of HT-TOFMS have been presented in previous publications [1][2][3]. Briefly, a continuous, accelerated ion beam is modulated on and off the axis of detection following a binary, pseudorandom encoding sequence that is applied at MHz rates. Because the produced ion packets have nanosecond widths, and typical flight times are on the order of tens or hundreds of microseconds, thousands of ion packets drift through the flight chamber at any given moment (compared to one packet in conventional TOFMS). Ions from these packets interpenetrate one another as they fly. Consequently, the detected signal corresponds to the s...

Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

Time-of-flight mass spectrometry (TOFMS) is a widely used technique that is recognized for offering high analytical performance at a reasonable cost. Development of this technique is ongoing, and advances in areas such as ion optics and ion-detection hardware have pushed the mass resolution and mass accuracy of TOFMS to regimes that are appropriate for the identification of components of complex mixtures.[1] The techniques intrinsic high ion transmission and capability to measure wide mass ranges without scanning, yields high sensitivity and fast spectral acquisition rates. Based on these characteristics, TOFMS seems to be an ideal detector for fast separations of analytes with a broad range of molecular weights.[2] Such applications, which include the inline separation of pharmaceuticals, peptides, or proteins followed by electrospray ionization, are becoming increasingly important.[3] Unfortunately, the pulsed nature of TOFMS yields inherent losses when analyzing ions emerging from continuous ion sources. Minimization of these losses can be achieved only at the expense of a reduction in the sampled mass range and potentially the mass resolution if the flight path is shortened. In conventional TOFMS, packets of ions are periodically pulsed into the entrance of a field-free drift chamber. To avoid overlap of the recorded flight times, the duration between start pulses is set to be longer than the flight time of the heaviest analyte ion. Ions reaching the entrance of the flight chamber between start pulses are lost. Thus, the ion sampling efficiency (duty cycle) and spectral acquisition speed are directly related to the ratio of the duration of the start pulse to the time between pulses, and these figures of merit decrease as the sampled mass range or flight path are increased.An ideal detector for capillary and chip-format separations should provide universal detection, sufficient spectral selectivity, and high sensitivity without degrading separation efficiency.[4] If TOFMS is to become the detector of choice for these applications, optimization of its transmission, speed, and efficiency is essential. One approach to improve the duty cycle is to modulate the continuous ion beam of a conventional TOF instrument to receive encoded single-ion packets, for example, by Fourier transform techniques.[5] The mass spectrum is then obtained by mathematical deconvolution and the analyzer duty cycle can be increased to about 25 %.The most widely used strategy for improving the duty cycle of TOFMS is orthogonal extraction (OE). [6] In this TOFMS configuration the fraction of the ion beam that is sampled is proportional to the length of the extraction region in the dimension orthogonal to the field-free flight trajectory. This region tends to be much larger than the sampling volume defined by ion gates used in an on-axis configuration. Thus, OE-TOFMS has a higher duty cycle than conventional onaxis TOFMS. But, because the flight times of ions traversing the extraction region depend on m/z, the duty cycle of OE-TOFMS decreases...