We introduce the TRUST4 open-source algorithm for reconstruction of immune receptor repertoires in αβ/γδ T cells and B cells from RNA-sequencing (RNA-seq) data. Compared with competing methods, ...TRUST4 supports both FASTQ and BAM format and is faster and more sensitive in assembling longer-even full-length-receptor repertoires. TRUST4 can also call repertoire sequences from single-cell RNA-seq (scRNA-seq) data without V(D)J enrichment, and is compatible with both SMART-seq and 5' 10x Genomics platforms.
High-throughput CRISPR screens have shown great promise in functional genomics. We present MAGeCK-VISPR, a comprehensive quality control (QC), analysis, and visualization workflow for CRISPR screens. ...MAGeCK-VISPR defines a set of QC measures to assess the quality of an experiment, and includes a maximum-likelihood algorithm to call essential genes simultaneously under multiple conditions. The algorithm uses a generalized linear model to deconvolute different effects, and employs expectation-maximization to iteratively estimate sgRNA knockout efficiency and gene essentiality. MAGeCK-VISPR also includes VISPR, a framework for the interactive visualization and exploration of QC and analysis results. MAGeCK-VISPR is freely available at http://bitbucket.org/liulab/mageck-vispr .
Chromatin immunoprecipitation, DNase I hypersensitivity and transposase-accessibility assays combined with high-throughput sequencing enable the genome-wide study of chromatin dynamics, transcription ...factor binding and gene regulation. Although rapidly accumulating publicly available ChIP-seq, DNase-seq and ATAC-seq data are a valuable resource for the systematic investigation of gene regulation processes, a lack of standardized curation, quality control and analysis procedures have hindered extensive reuse of these data. To overcome this challenge, we built the Cistrome database, a collection of ChIP-seq and chromatin accessibility data (DNase-seq and ATAC-seq) published before January 1, 2016, including 13 366 human and 9953 mouse samples. All the data have been carefully curated and processed with a streamlined analysis pipeline and evaluated with comprehensive quality control metrics. We have also created a user-friendly web server for data query, exploration and visualization. The resulting Cistrome DB (Cistrome Data Browser), available online at http://cistrome.org/db, is expected to become a valuable resource for transcriptional and epigenetic regulation studies.
Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for ...the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires ~3 h on a desktop computer running Linux or Mac OS with R support.
RNA-seq has been widely used in transcriptome analysis to effectively measure gene expression levels. Although sequencing costs are rapidly decreasing, almost 70% of all the human RNA-seq samples in ...the gene expression omnibus do not have biological replicates and more unreplicated RNA-seq data were published than replicated RNA-seq data in 2011. Despite the large amount of single replicate studies, there is currently no satisfactory method for detecting differentially expressed genes when only a single biological replicate is available.
We present the GFOLD (generalized fold change) algorithm to produce biologically meaningful rankings of differentially expressed genes from RNA-seq data. GFOLD assigns reliable statistics for expression changes based on the posterior distribution of log fold change. In this way, GFOLD overcomes the shortcomings of P-value and fold change calculated by existing RNA-seq analysis methods and gives more stable and biological meaningful gene rankings when only a single biological replicate is available.
The open source C/C++ program is available at http://www.tongji.edu.cn/∼zhanglab/GFOLD/index.html
Endocrine therapies for breast cancer that target the estrogen receptor (ER) are ineffective in the 25%–30% of cases that are ER negative (ER–). Androgen receptor (AR) is expressed in 60%–70% of ...breast tumors, independent of ER status. How androgens and AR regulate breast cancer growth remains largely unknown. We find that AR is enriched in ER– breast tumors that overexpress HER2. Through analysis of the AR cistrome and androgen-regulated gene expression in ER–/HER2+ breast cancers we find that AR mediates ligand-dependent activation of Wnt and HER2 signaling pathways through direct transcriptional induction of WNT7B and HER3. Specific targeting of AR, Wnt or HER2 signaling impairs androgen-stimulated tumor cell growth suggesting potential therapeutic approaches for ER–/HER2+ breast cancers.
► AR is highly expressed in ER–/HER2+ breast tumors ► Androgen and AR stimulate the growth of ER–/HER2+ breast cancer cells ► AR activates Wnt and HER2 pathways by transcriptional induction of
WNT7B and
HER3 ► Targeting AR effectively inhibits the growth of ER–/HER2+ breast tumors
According to the LM22 matrix, the expression levels are highly correlated (r = 0.9). In the original manuscript 1, we did not make arguments regarding the estimation of the absolute proportion of a ...single cell type. ...we are surprised by the claim by Newman et al. ...we were fully aware that TIMER estimates may correlate with total leukocyte levels (Fig. ...in the downstream analyses, we used the partial Spearman correlation conditioning on tumor purity whenever necessary to control for this factor. ...we would like to re-emphasize that CIBERSORT and TIMER target different aspects of tumor immune infiltrates.
Next-generation sequencing (NGS) technologies have been used in diverse ways to investigate various aspects of chromatin biology by identifying genomic loci that are bound by transcription factors, ...occupied by nucleosomes or accessible to nuclease cleavage, or loci that physically interact with remote genomic loci. However, reaching sound biological conclusions from such NGS enrichment profiles requires many potential biases to be taken into account. In this Review, we discuss common ways in which biases may be introduced into NGS chromatin profiling data, approaches to diagnose these biases and analytical techniques to mitigate their effect.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation ...(CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.
Cancer immunogenomics originally was framed by research supporting the hypothesis that cancer mutations generated novel peptides seen as “non-self” by the immune system. The search for these ...“neoantigens” has been facilitated by the combination of new sequencing technologies, specialized computational analyses, and HLA binding predictions that evaluate somatic alterations in a cancer genome and interpret their ability to produce an immune-stimulatory peptide. The resulting information can characterize a tumor’s neoantigen load, its cadre of infiltrating immune cell types, the T or B cell receptor repertoire, and direct the design of a personalized therapeutic.
The development of new high-throughput technologies facilitates the application of immunogenomics to cancer.
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