2019 Nocturnin RNA-seq analysis code

Overview

This is the repository holding all formatted scripts used in bioinformatics analysis for:

Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide dinucleotide metabolites Elizabeth T. Abshire, Kelsey L. Hughes, Rucheng Diao, Sarah Pearce, Raymond C. Trievel, Joanna Rorbach, Peter L. Freddolino, Aaron C. Goldstrohm bioRxiv 2020.01.12.903534; doi: https://doi.org/10.1101/2020.01.12.903534

Detailed methods and statistics are described in the Materials and Methods in the paper. Briefly, raw RNA-seq data was quality controlled and sequencing reads were trimming accordingly. The data was then aligned to a custom reference genome containing human reference genome, ERCC, and overexpression components. The alignment was quantified and analyzed for differential expression and functional enrichment.

In all codes, the user name of submitter/analyzer has been replaced by string user, and the user name of the principle investigator has been replaced by string PI.

List of scripts

General:

• LICENSE;
• README.md

Patch to existing tools:

• DESeq2.1.61.1.patch.R: patched function applied to DESeq2 (version 1.16.1) to include lfcSE in the shrinkage output

Common environmental variables and paths:

• noct-00-envSetup.sh: script to set paths to scripts and tools used in analysis, define global environmental variable and reused variables

Quality control:

• noct-00-linkFastq.sh: script to create symbolic links at location of data analysis directory to raw sequencing data;
• noct-01-qc.sh: script to perform quality control on raw sequencing reads

Read sequence processing:

• noct-02-cutAndTrim.sh
• noct-02-qc.sh

Alignment and quantification:

• noct-03-pipeline-runStarHtseq.sh: pipelien script running STAR and HTSeq for alignment and quantification by calling noct-03-runStar.sh and noct-04-quantHTSeq.sh;
• noct-03-buildStarRefGTF.py: Python3 script to build annotation (GTF files) and sequences (FASTA) information for ERCC spike-ins and components of over-expression vectors;
• noct-03-runStar.sh: script to build STAR reference and run STAR alignment;
• noct-04-quantHTSeq.sh: script to run HTSeq quantification

Differential gene expression analysis:

• noct-05-deseq2.htseq.R: script to analyze HTSeq quantification results with DESeq2 for differential expression;
• noct-05-targetConfIntv.R: script to calculate target confidence intervals for Figure 5d

GO term analysis:

• noct-06-ipageDb.R: script to query and build database files used in iPAGE analysis;
• noct-06-calcZLFC.R: script to calculate z-scores of log2-fold-changes based on DESeq2 results, as input for iPAGE;
• noct-06-ipage.sh: commands for iPAGE calls

Visualization and table summary (in the order of reference in manuscript):

• noct-07-ma.R: script to plot MA plot (Figure 5b);
• noct-07-plotVolcanoGoGene.R : scirpt to plot volcano plot (Figure 5c);
• noct-07-plotGoTerm.R: script to plot GO-term-specific gene expression level bar charts (Supporting Information Figure S5 - S8);
• noct-07-mitoCategory.R: script to add mitochondria-related categorization to TPM table (Table S1, see below) for further visualization as notched box plot (Fig S9, see below);
• noct-07-notchedBox.R: script to plot notched boxplot for expression change levels of each mitochondria-related ccategory (Supporting Information Figure S9);
• noct-07-totalExonLength.R: script to query and calculate the total exon length for each gene for normalization in TPM calculation (noct-07-tpm.R);
• noct-07-tpm.R: calculate the TPM of gene expression (Supporting Information Table S1);
• noct-07-ipageTable.R: summarize significant GO terms by iPAGE as a .csv file (Supporting Information Table S3)

Figure 6 (GO term analysis) is the iPAGE output PDF file reformatted only for presentation styles. Therefore no script is listed above for Figure 6.