A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila, bioRxiv, 2019-05-13

SUMMARYGenetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1400 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

biorxiv genetics 100-200-users 2019

Determining sufficient sequencing depth in RNA-Seq differential expression studies, bioRxiv, 2019-05-13

AbstractRNA-Seq studies require a sufficient read depth to detect biologically important genes. Sequencing below this threshold will reduce statistical power while sequencing above will provide only marginal improvements in power and incur unnecessary sequencing costs. Although existing methodologies can help assess whether there is sufficient read depth, they are unable to guide how many additional reads should be sequenced to reach this threshold. We provide a new method called superSeq that models the relationship between statistical power and read depth. We apply the superSeq framework to 393 RNA-Seq experiments (1,021 total contrasts) in the Expression Atlas and find the model accurately predicts the increase in statistical power gained by increasing the read depth. Based on our analysis, we find that most published studies (> 70%) are undersequenced, i.e., their statistical power can be improved by increasing the sequencing read depth. In addition, the extent of saturation is highly dependent on statistical methodology only 9.5%, 29.5%, and 26.6% of contrasts are saturated when using DESeq2, edgeR, and limma, respectively. Finally, we also find that there is no clear minimum per-transcript read depth to guarantee saturation for an entire technology. Therefore, our framework not only delineates key differences among methods and their impact on determining saturation, but will also be needed even as technology improves and the read depth of experiments increases. Researchers can thus use superSeq to calculate the read depth to achieve required statistical power while avoiding unnecessary sequencing costs.

biorxiv genomics 100-200-users 2019

 

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