RNA-Seq uses next-gen sequencing to show the presence and quantity of RNA in a genome at a particular moment. DNASTAR’s SeqMan NGen application is the starting point for both reference-guided and de novo RNA-Seq workflows. Because this tutorial involves a reference-guided workflow, you will then use ArrayStar to analyze the completed RNA-Seq assembly.
In this tutorial, you will compare stationary phase RNA from wild-type E. coli cells with that from two different mutant cells, each lacking one of two genes that together encode a transcription factor for flagella and chemotaxis operons. An “operon” is a group of one or more genes that are transcribed as a single RNA unit.
In Part A, you will use SeqMan NGen to create a reference-guided DESeq2-normalized RNA-Seq assembly for two experimental replicates and one wild type control of E. coli transcription factors FlhC and FlhD. The data you will assemble is publicly-available single-end Illumina data from E. coli (Fitzgerald et al, 2014). In Parts B & C, you will use ArrayStar to perform downstream analysis and will learn to identify flagella-related operons using two different techniques.
IMPORTANT! There are two options for following this tutorial, one significantly longer than the other.
|Item||Full tutorial||Abbreviated tutorial|
|Description||Assemble in SeqMan NGen, then do downstream analysis in ArrayStar||Do only the downstream analysis in ArrayStar, using a provided project|
|Download size||4.0 GB (unzips to 16 GB)||4.3 MB|
|Assembly wait time||1-3 hours||none|
|Where to begin||Perform the steps in Part A: Setting up the RNA-Seq reference-guided assembly in SeqMan NGen||Read the steps in Part A: Setting up the RNA-Seq reference-guided assembly in SeqMan NGen without performing them|
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