Methods to study splicing from high-throughput RNA Sequencing data

The development of novel high-throughput sequencing (HTS) methods for RNA (RNA-Seq) has provided a very powerful mean to study splicing under multiple conditions at unprecedented depth. However, the complexity of the information to be analyzed has turned this into a challenging task. In the last few years, a plethora of tools have been developed, allowing researchers to process RNA-Seq data to study the expression of isoforms and splicing events, and their relative changes under different conditions. We provide an overview of the methods available to study splicing from short RNA-Seq data. We group the methods according to the different questions they address: 1) Assignment of the sequencing reads to their likely gene of origin. This is addressed by methods that map reads to the genome and/or to the available gene annotations. 2) Recovering the sequence of splicing events and isoforms. This is addressed by transcript reconstruction and de novo assembly methods. 3) Quantification of events and isoforms. Either after reconstructing transcripts or using an annotation, many methods estimate the expression level or the relative usage of isoforms and/or events. 4) Providing an isoform or event view of differential splicing or expression. These include methods that compare relative event/isoform abundance or isoform expression across two or more conditions. 5) Visualizing splicing regulation. Various tools facilitate the visualization of the RNA-Seq data in the context of alternative splicing. In this review, we do not describe the specific mathematical models behind each method. Our aim is rather to provide an overview that could serve as an entry point for users who need to decide on a suitable tool for a specific analysis. We also attempt to propose a classification of the tools according to the operations they do, to facilitate the comparison and choice of methods.Comment: 31 pages, 1 figure, 9 tables. Small corrections adde

Science with a small two-band UV-photometry mission II: Observations of stars and stellar systems

We outline the impact of a small two-band UV-photometry satellite mission on the field of stellar physics, magnetospheres of stars, binaries, stellar clusters, interstellar matter, and exoplanets. On specific examples of different types of stars and stellar systems, we discuss particular requirements for such satellite missions in terms of specific mission parameters such as bandpass, precision, cadence, and mission duration. We show that such a mission may provide crucial data not only for hot stars that emit most of their light in UV, but also for cool stars, where UV traces their activity. This is important, for instance, for exoplanetary studies, because the level of stellar activity influences habitability. While the main asset of the two-band UV mission rests in time-domain astronomy, an example of open clusters proves that such a mission would be important also for the study of stellar populations. Properties of the interstellar dust are best explored when combining optical and IR information with observations in UV. It is well known that dust absorbs UV radiation efficiently. Consequently, we outline how such a UV mission can be used to detect eclipses of sufficiently hot stars by various dusty objects and study disks, rings, clouds, disintegrating exoplanets or exoasteroids. Furthermore, UV radiation can be used to study the cooling of neutron stars providing information about the extreme states of matter in the interiors of neutron stars and used for mapping heated spots on their surfaces.Comment: Submitted to Space Science Review

A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

BackgroundOncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance.ResultsIn reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5-100x more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels.ConclusionThese new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.Peer reviewe

Evaluation of reactive power meters in the presence of high harmonic distortion

NRC publication: Ye

Comparison of enclosed space detection system with conventional methods

Enclosed Space Detection System (ESDS) is a fast, inexpensive, and reliable device for detecting human occupants hidden in vehicles. Operation requires less than two minutes. ESDS is used to foil attempts at smuggling illegal aliens, terrorists, and escaping prisoners. It is being tested at nuclear weapons facilities and has been operated at several prisons and international border crossings. ESDS is the first practical electronic alternative to physical searches of vehicles for hidden passengers. At critical checkpoints, a thorough physical search of a single fully loaded truck requires a team of from two to six people, and may take as long as eight hours. Despite this level of security, experience has shown that the search can occasionally be foiled. Due to the enormous time and expense of thorough physical searches of vehicles, they are seldom conducted at any but the most critical of locations, simply leaving many sites vulnerable to crime and terrorism. Prior to the development of the ESDS, the only other effective alternative to physical search was the use of specially-trained canines, which can be vastly superior to the physical search in both time and accuracy. However, as discussed in this paper, canine inspection is not really a competitive substitute for ESDS because canine reliability (80% at most) is not as high as that of the ESDS (99%+), while the costs, training requirements, and operator skill needed are significantly higher with canines than with the ESDS. In addition, the ESDS has straightforward self-diagnostic tests to ensure the system is operating correctly; such tests are not currently available with either canine or human inspectors. ESDS offers an attractive supplement or alternative to meet current security requirements for vehicle searches at portals at government, nuclear, industrial, and other facilities where concealed persons may pose a threat either by entering or leaving

Application of the smart portal in transportation

Under a program sponsored by the Department of Energy, the Oak Ridge complex is developed a ``Portal-of-the-Future``, or ``smart portal``. This is a security portal for vehicular traffic which is intended to quickly detect explosives, hidden passengers, etc. It uses several technologies, including microwaves, weigh-in-motion, digital image processing, and electroacoustic wavelet-based heartbeat detection. A novel component of particular interest is the Enclosed Space Detection System (ESDS), which detects the presence of persons hiding in a vehicle. The system operates by detecting the presence of a human ballistocardiographic signature. Each time the heart beats, it generates a small but measurable shock wave that propagates through the body. The wave, whose graph is called a ballistocardiogram, is the mechanical analog of the electrocardiogram, which is routinely used for medical diagnosis. The wave is, in turn, coupled to any surface or object with which the body is in contact. If the body is located in an enclosed space, this will result in a measurable deflection of the surface of the enclosure. Independent testing has shown ESDS to be highly reliable. The technologies used in the smart portal operate in real time and allow vehicles to be checked through the portal in much less time than would be required for human inspection. Although not originally developed for commercial transportation, the smart portal has the potential to solve several transportation problems. It could relieve congestion at international highway border crossings by reducing the time required to inspect each vehicle while increasing the level of security. It can reduce highway congestion at the entrance of secure facilities such as prisons. Also, it could provide security at intermodal transfer points, such as airport parking lots and car ferry terminals

The SEQC2 epigenomics quality control (EpiQC) study

Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group. Results Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. Conclusions The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.Correction in: Genome Biology, Volume 22, Issue 1, Article Number 350, DOI 10.1186/s13059-021-02573-y</p

A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium

We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the US Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed for all examined platforms, including qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings