Biology subjects

Yin, P.

Publications and source records attributed to Yin, P..

6 recordsLinked to original sources

bioRxiv2018

SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

Fluorescent in situ hybridization (FISH) reveals the abun-dance and positioning of nucleic acid sequences in fixed sam-ples and can be combined with cell segmentation to produce a powerful single cell gene expression assay. However, it re-mains difficult to label more than a few targets and to visu-alize nucleic acids in environments such as thick tissue sam-ples using conventional FISH technologies. Recently, meth-ods have been developed for multiplexed amplification of FISH signals, yet it remains challenging to achieve high lev-els of simultaneous multiplexing combined with high sam-pling efficiency and simple workflows. Here, we introduce signal amplification by exchange reaction (SABER), which endows oligo-based FISH probes with long, single-stranded DNA concatemers that serve as targets for sensitive fluores-cent detection. We establish that SABER effectively ampli-fies the signal of probes targeting nucleic acids in fixed cells and tissues, can be deployed against at least 17 targets si-multaneously, and detects mRNAs with high efficiency. As a demonstration of the utility of SABER in assays involv-ing genetic manipulations, we apply multiplexed FISH of reporters and cell type markers to the identification of en-hancers with cell type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit to allow rapid and cost effective multiplexed imaging.

genetics↗

bioRxiv2018

Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

Chromosome structure is thought to be crucial for proper functioning of the nucleus. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. We begin by applying Oligopaint probes and the single-molecule localization microscopy methods of OligoSTORM and OligoDNA-PAINT to image 8 megabases of human chromosome 19, discovering that chromosomal regions contributing to compartments can form distinct structures. Intriguingly, our data also suggest that homologous maternal and paternal regions may be differentially organized. Finally, we integrate imaging data with Hi-C and restraint-based modeling using a method called integrative modeling of genomic regions (IMGR) to increase the genomic resolution of our traces to 10 kb.\n\nOne Sentence SummarySuper-resolution genome tracing, contact maps, and integrative modeling enable 10 kb resolution glimpses of chromosome folding.

genetics↗

bioRxiv2017

Task Engagement Enhances Population Encoding of Stimulus Meaning in Primary Auditory Cortex

The main functions of primary sensory cortical areas are classically considered to be the extraction and representation of stimulus features. In contrast, higher cortical sensory association areas are thought to be responsible for combining these sensory representations with internal motivations and learnt associations. These regions generate appropriate neural responses that are maintained until a motor command is executed. Within this framework, responses of the primary sensory areas during task performance are expected to carry less information about the behavioral meaning of the stimulus than higher sensory, association, motor and frontal cortices. Here we demonstrate instead that the neuronal population responses in the early primary auditory cortex (A1) display many aspects of responses generally associated with higher-level areas. A1 activity was recorded in awake ferrets while they were either passively listening or actively discriminating two periodic click trains of different rates in a Go/No-Go paradigm. By applying population-level dimensionality reduction techniques, we found that task-engagement induced a shift in the nature of the encoding from a sensory-driven representation of the two stimuli to a behaviorally relevant representation of the two categories that specifically enhances the target stimulus. We demonstrate that this shift in encoding relies partly on a novel mechanism of change in spontaneous activity patterns upon engagement in the task. We show that this population-level representation of stimuli in A1 population activity bears strong similarities to responses in the frontal cortex, but appears earlier following stimulus presentation. Analysis of neural activity recorded in various Go/No-Go tasks, with different sounds and reinforcement paradigms, reveals that this striking population-level enhancement of target representation is a general property of task engagement. These findings indicate that primary sensory cortices play a highly flexible role in the processing of incoming stimuli and implement a crucial change in the structure of population activity in order to extract task-relevant information during behavior.

neuroscience↗

bioRxiv2017

MeDEStrand: an improved method to infer genome-wide absolute methylation level from DNA enrichment experiment

BackgroundDNA methylation of dinucleotide CpG is an essential epigenetic modification that plays a key role in transcription. Bisulfite conversion method is a \"gold standard\" for DNA methylation profiling that provides single nucleotide resolution. However, whole-genome bisulfite conversion is very expensive. Alternatively, DNA enrichment-based methods offer high coverage of methylated CpG dinucleotides with the lowest cost per CpG covered genome-wide and have been used widely. They measure the DNA enrichment of methyl-CpG binding, therefore do not directly provide absolute methylation levels. Further, the enrichment is influenced by confounding factors besides the methylation status, e.g., CpG density. Computational models that can accurately derive the absolute methylation levels from the enrichment data are necessary.\n\nResultsWe present MeDEStrand, a method uses sigmoid function to estimate and correct the CpG bias from the numbers of reads that fell within bins that divide the genome. In addition, unlike the previous methods, which estimate CpG bias based on reads mapped at the same genomic loci, MeDEStrand processes the reads for the positive and negative DNA strands separately. We compare the performance of MeDEStrand with three other state-of-the-art methods MEDIPS, BayMeth and QSEA on four independent datasets generated using immortalized cell lines (GM12878 and K562) and human patient primary cells (foreskin fibroblast and mammary epithelial). Based on the comparison between the inferred absolute methylation levels from MeDIP-seq and the corresponding RRBS data, MeDEStrand shows the best performance at high resolution of 25, 50 and 100 base pairs.\n\nConclusions MeDEStrand benefits from the estimation of CpG bias with a sigmoid function and the procedure to process reads mapped to the positive and negative DNA strands separately. MeDEStrand is a tool to infer whole-genome absolute DNA methylation level at the cost of enrichment-based methods with adequate accuracy and resolution. R package MeDEStrand and its tutorial is freely available for download at https://github.com/jxu1234/MeDEStrand.git

bioinformatics↗

bioRxiv2017

OligoMiner: A rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes

Oligonucleotide (oligo)-based fluorescence in situ hybridization (FISH) has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate existing and new utilities into the pipeline as desired, and introduces a novel method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with both diffraction-limited and single-molecule super-resolution imaging of chromosomal and RNA targets. We anticipate this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.

bioinformatics↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗