Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression, bioRxiv, 2019-03-14

AbstractSingle-cell RNA-seq (scRNA-seq) data exhibits significant cell-to-cell variation due to technical factors, including the number of molecules detected in each cell, which can confound biological heterogeneity with technical effects. To address this, we present a modeling framework for the normalization and variance stabilization of molecular count data from scRNA-seq experiments. We propose that the Pearson residuals from ’regularized negative binomial regression’, where cellular sequencing depth is utilized as a covariate in a generalized linear model, successfully remove the influence of technical characteristics from downstream analyses while preserving biological heterogeneity. Importantly, we show that an unconstrained negative binomial model may overfit scRNA-seq data, and overcome this by pooling information across genes with similar abundances to obtain stable parameter estimates. Our procedure omits the need for heuristic steps including pseudocount addition or log-transformation, and improves common downstream analytical tasks such as variable gene selection, dimensional reduction, and differential expression. Our approach can be applied to any UMI-based scRNA-seq dataset and is freely available as part of the R package sctransform, with a direct interface to our single-cell toolkit Seurat.

biorxiv genomics 200-500-users 2019

Frequent birth of de novo genes in the compact yeast genome, bioRxiv, 2019-03-13

AbstractEvidence has accumulated that some genes originate directly from previously non-genic sequences, or de novo, rather than by the duplication or fusion of existing genes. However, how de novo genes emerge and eventually become functional is largely unknown. Here we perform the first study on de novo genes that uses transcriptomics data from eleven different yeast species, all grown identically in both rich media and in oxidative stress conditions. The genomes of these species are densely-packed with functional elements, leaving little room for the co-option of genomic sequences into new transcribed loci. Despite this, we find that at least 213 transcripts (~5%) have arisen de novo in the past 20 million years of evolution of baker’s yeast-or approximately 10 new transcripts every million years. Nearly half of the total newly expressed sequences are generated from regions in which both DNA strands are used as templates for transcription, explaining the apparent contradiction between the limited ‘empty’ genomic space and high rate of de novo gene birth. In addition, we find that 40% of these de novo transcripts are actively translated and that at least a fraction of the encoded proteins are likely to be under purifying selection. This study shows that even in very highly compact genomes, de novo transcripts are continuously generated and can give rise to new functional protein-coding genes.

biorxiv evolutionary-biology 0-100-users 2019

Frequent birth ofde novogenes in the compact yeast genome, bioRxiv, 2019-03-13

AbstractEvidence has accumulated that some genes originate directly from previously non-genic sequences, orde novo, rather than by the duplication or fusion of existing genes. However, howde novogenes emerge and eventually become functional is largely unknown. Here we perform the first study onde novogenes that uses transcriptomics data from eleven different yeast species, all grown identically in both rich media and in oxidative stress conditions. The genomes of these species are densely-packed with functional elements, leaving little room for the co-option of genomic sequences into new transcribed loci. Despite this, we find that at least 213 transcripts (~5%) have arisende novoin the past 20 million years of evolution of baker’s yeast-or approximately 10 new transcripts every million years. Nearly half of the total newly expressed sequences are generated from regions in which both DNA strands are used as templates for transcription, explaining the apparent contradiction between the limited ‘empty’ genomic space and high rate ofde novogene birth. In addition, we find that 40% of thesede novotranscripts are actively translated and that at least a fraction of the encoded proteins are likely to be under purifying selection. This study shows that even in very highly compact genomes,de novotranscripts are continuously generated and can give rise to new functional protein-coding genes.

biorxiv evolutionary-biology 0-100-users 2019

 

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