UNLABELLED: RNA-seq, the application of next-generation sequencing to RNA, provides transcriptome-wide characterization of cellular activity. Assessment of sequencing performance and library quality is critical to the interpretation of RNA-seq data, yet few tools exist to address this issue. We introduce RNA-SeQC, a program which provides key measures of data quality. These metrics include yield, alignment and duplication rates; GC bias, rRNA content, regions of alignment (exon, intron and intragenic), continuity of coverage, 3'/5' bias and count of detectable transcripts, among others. The software provides multi-sample evaluation of library construction protocols, input materials and other experimental parameters. The modularity of the software enables pipeline integration and the routine monitoring of key measures of data quality such as the number of alignable reads, duplication rates and rRNA contamination. RNA-SeQC allows investigators to make informed decisions about sample inclusion in downstream analysis. In summary, RNA-SeQC provides quality control measures critical to experiment design, process optimization and downstream computational analysis. AVAILABILITY AND IMPLEMENTATION: See www.genepattern.org to run online, or www.broadinstitute.org/rna-seqc/ for a command line tool.

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (< 1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved > 97% sensitivity and > 97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the -10 kb genome, including 603 rare variants (< 1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses.

Most man-made objects can be closely approximated by bodies whose surfaces are composed of portions of second-order (quadric) surfaces. These surfaces include elliptic, hyperbolic, and parabolic cylinders, as well as quadric cones, paraboloids, hyperboloids, ellipsoids, and pairs of planes. Simple planes (first-order surfaces) may be included as degenerate quadric surfaces.Because these quadric-surface bodies are so useful for modelling man-made objects, it is important that any Computer-Aided Design (CAD) system be able to work with such bodies. The “QUADRIL” language described here was designed to accept descriptions of quadric-surface bodies in character-string form.QUADRIL has a mixture of English-like and algebraic syntax. It may be used to specify quadric-surface bodies and then to display them on various media.QUADRIL will accept descriptions of quadric-surface bodies either as “volumetric” combinations of basic bodies, or as boolean functions of bounding surfaces. English-like syntax is used for specifying what surfaces and basic bodies are used, while algebraic syntax is used to transform the canonical forms of the surfaces or bodies into the shape, position, and orientation that the user desires.Volumetric combination of bodies involves the operations of union (+), intersection (*), and subtraction (-). Boolean specification of volumes is in terms of a boolean tree with the bounding surfaces as leaf nodes. The tree is expressed as a character string.QUADRIL permits using user-created “STRUCTURES” as component bodies (“OBJECTS”) in greater STRUCTURES.The display of the quadric-surface bodies may also be specified in QUADRIL. The user is considered fixed in space, while the body is transformed to give the desired view.

An algorithm for drawing pictures of three-dimensional objects, with surfaces made up of patches of quadric surfaces, is described.The emphasis of this algorithm is calculating the intersections of quadric surfaces. A parameterization scheme is used. Each quadric surface intersection curve (QSIC) is represented as a set of coefficients and parameter limits. Each value of the parameter represents at most two points, and these may easily be distinguished. This scheme can find the co-ordinates of points of even quartic (fourth-order) intersection curves, using equations of no more than second order.Methods of parameterization for each type of QSIC are discussed, as well as surface bounding and hidden surface removal.