Detecting simultaneous variant intervals in aligned sequences

Given a set of aligned sequences of independent noisy observations, we are concerned with detecting intervals where the mean values of the observations change simultaneously in a subset of the sequences. The intervals of changed means are typically short relative to the length of the sequences, the subset where the change occurs, the "carriers," can be relatively small, and the sizes of the changes can vary from one sequence to another. This problem is motivated by the scientific problem of detecting inherited copy number variants in aligned DNA samples. We suggest a statistic based on the assumption that for any given interval of changed means there is a given fraction of samples that carry the change. We derive an analytic approximation for the false positive error probability of a scan, which is shown by simulations to be reasonably accurate. We show that the new method usually improves on methods that analyze a single sample at a time and on our earlier multi-sample method, which is most efficient when the carriers form a large fraction of the set of sequences. The proposed procedure is also shown to be robust with respect to the assumed fraction of carriers of the changes.Comment: Published in at http://dx.doi.org/10.1214/10-AOAS400 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

Genome analysis: mutation analysis using near infrared laser-induced fluorescence (NIR-LIF) and single molecule detection in microfluidic devices

A number of genotyping methods have been developed for mutation analysis, each of which has its own unique advantage. DNA amplification via polymerase chain reaction (PCR) provides an unlimited supply of material for subsequent genetic analysis even in case where only a single copy of the DNA molecule is present in the sample. Research presented in this dissertation first examines the efficiency of in vitro amplification of single copy DNA with subsequent sequencing analysis of PCR product. The PCR products (amplicons) were investigated for possible alteration, distortions or mutations due to the amplification process. The sequencing data for single copy amplification indicated a read length of 424 bases could be achieved with read accuracy of 99.3%. A confocal detection system was constructed to monitor the fluorescence signature from single DNA molecule labeled with near infrared (NIR) dyes. The performance of the system was first tested by applying single photon burst technique to detect double-stranded DNA molecules in polymeric devices. Sampling efficiencies were investigated by physically narrowing the channel sizes of the microdevices and by applying electrokinetic focusing. The narrow channels showed 4 times improvement in a 15 µm channel compared to a 50 µm channel. Similar results were demonstrated in the focusing studies. Single molecule sizing of ë-DNA, M13, pUC19 and pBR322 DNA using single photon burst was also demonstrated. In addition single molecule detection of a NIR chromophore NN383 was analyzed in PC devices and a detection efficiency of 94% was achieved. The other application for the NIR system was in a new strategy for analyzing molecular signatures of disease states in real-time using single-pair fluorescence energy transfer (spFRET) coupled with ligase detection reaction (LDR) to rapidly detect single base mutations in codon 12 of K-ras oncogene which has high diagnostic value for colorectal cancer. LDR-spFRET provided the necessary specificity and sensitivity to detect single point mutations in as little 600 copies of human genomic DNA without PCR amplification at a level of 1 in 1000 wildtype sequences. In addition the assay demonstrated analysis times \u3c 5 min

CNARA: reliability assessment for genomic copy number profiles

BACKGROUND DNA copy number profiles from microarray and sequencing experiments sometimes contain wave artefacts which may be introduced during sample preparation and cannot be removed completely by existing preprocessing methods. Besides, large derivative log ratio spread (DLRS) of the probes correlating with poor DNA quality is sometimes observed in genome screening experiments and may lead to unreliable copy number profiles. Depending on the extent of these artefacts and the resulting misidentification of copy number alterations/variations (CNA/CNV), it may be desirable to exclude such samples from analyses or to adapt the downstream data analysis strategy accordingly. RESULTS Here, we propose a method to distinguish reliable genomic copy number profiles from those containing heavy wave artefacts and/or large DLRS. We define four features that adequately summarize the copy number profiles for reliability assessment, and train a classifier on a dataset of 1522 copy number profiles from various microarray platforms. The method can be applied to predict the reliability of copy number profiles irrespective of the underlying microarray platform and may be adapted for those sequencing platforms from which copy number estimates could be computed as a piecewise constant signal. Further details can be found at https://github.com/baudisgroup/CNARA . CONCLUSIONS We have developed a method for the assessment of genomic copy number profiling data, and suggest to apply the method in addition to and after other state-of-the-art noise correction and quality control procedures. CNARA could be instrumental in improving the assessment of data used for genomic data mining experiments and support the reliable functional attribution of copy number aberrations especially in cancer research

Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging

Both multi-cell biochemical assays and single cell fluorescence measurements have revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Employing high-speed 3D fluorescence nanoimaging we resolve here at the single cell level multiple, distinct regions of mRNA synthesis within a labeled transgene array. By employing phasor analysis, a fluorescence fluctuation spectroscopy technique, we demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm, and we extract the local PolII elongation rate. While we detect a range of 10-100 bp/s for PolII elongation from cell to cell, we are now also able to measure up to a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell. Furthermore, we are able to visualize changes of PolII elongation as a function of time. We observe a correlation between the average elongation rate measured in a locus and its local mobility. Finally, by cross-correlating the transcriptional activity with the nm-sized movements of the active loci, we provide evidence of an active molecular mechanism determining displacements of the transcription sites concomitant to increases in transcriptional activity. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability.Comment: 56 pages, 5 main figures and 10 supplementary figure

Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20\u2013250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus

Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry

AbstractFluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used previously to determine the stoichiometry of small oligomers in cells. We extend brightness analysis to large macromolecular protein complexes containing thousands of proteins and determine their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to determine the stoichiometry of HIV-1 Gag within the particles. Control experiments establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The experiments further show that the brightness scales linearly with the amount of labeled Gag within the particle. Brightness analysis shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not constant, but varies with the amount of transfected DNA plasmid. We observed HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP production. This study shows that brightness analysis has the potential to become an important tool for investigating large molecular complexes by providing quantitative information about their size and composition