High-resolution Expression Profiling of Selected Gene Sets during Plant Immune Activation
Bruno Pok Man Ngou,
Oliver J. Furzer,
Ram Krishna Shrestha,
Jonathan DG Jones
Posted 23 Sep 2019
bioRxiv DOI: 10.1101/775973 (published DOI: 10.1111/pbi.13327)
Posted 23 Sep 2019
Sequence capture followed by next-generation sequencing has broad applications in cost-effective exploration of biological processes at high resolution. Genome-wide RNA sequencing (RNA-seq) over a time course can reveal the dynamics of differential gene expression. However, in many cases, only a limited set of genes are of interest, and are repeatedly used as markers for certain biological processes. Sequence capture can help generate high-resolution quantitative datasets to assess changes in abundance of selected genes. We previously used sequence capture to accelerate Resistance gene cloning, investigate immune receptor gene diversity and investigate pathogen diversity and evolution. The plant immune system involves detection of pathogens via both cell-surface and intracellular receptors. Both receptor classes can induce transcriptional reprogramming that elevates disease resistance. To assess differential gene expression during plant immunity, we developed and deployed quantitative sequence capture (CAP-I). We designed and synthesized biotinylated single-strand RNA bait libraries targeted to a subset of defense genes, and generated sequence capture data from 99 RNA-seq libraries. We built a data processing pipeline to quantify the RNA-CAP-I-seq data, and visualize differential gene expression. Sequence capture in combination with quantitative RNA-seq enabled cost-effective assessment of the expression profile of a specified subset of genes. Quantitative sequence capture is not limited to RNA-seq or any specific organism and can potentially be incorporated into automated platforms for high-throughput sequencing.
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