pevs / SMRI_ms_ACC

Scripts and overview of project to analyze RNA-seq data from anterior cingulate cortex in schizophrenia

SMRI_Array_ACC_SZ

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Description

Analysis of RNA-Seq from the anterior cingulate cortex (ACC) of patients with schizophrenia (SZ) and unaffected controls (CTRL), from the Stanley Medical Research Institute (SMRI) Array Collection, using qSVA methodology to adjust for postmortem RNA degradation and derfinder for flexible expression quantification. Results were compared with RNA-Seq data from prefrontal cortex (PFC) and hippocampus (HPC) of the same individuals.

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Data

Anterior cingulate cortex (ACC; BA24)

Zhao, et al. Transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder. Mol Psych 2015. Data available from SMRI.

Hippocampus (HPC)

Darby, et al. Consistently altered expression of gene sets in postmortem brains of individuals with major psychiatric disorders. Transl Psych 2016. Data available from SMRI.

Dorsolateral pre-frontal cortex (PFC; BA46)

PsychENCODE's BrainGVEx. Data available from PsychENCODE Consortium’s BrainGVEX project.

Non-RNA-Seq

Additional data (e.g., qRT-PCR, protein expression, and cell type counts) available for samples at SMRI Database.

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Analysis workflow

1. Alignment

• run_align_hisat2.sh

2. Quantify feature expression

Genes, exons, and junctions (featureCounts)

• run_counts_genes_exons_jxns.sh
• run_anno_genes_exons.R
• run_anno_jxns.R

Regions (derfinder)

• run_counts_regions.sh
• run_anno_regions.sh

3. Preprocessing

Library size and degradation stats

• libsize_degstats.R

Combine region datasets

• exprsData_import_genes_exons.R
• exprsData_import_jxns.R
• exprsData_import_regions.R
• hpc_fix_qsva_regions.R

Filtering samples and features

• exprsData_prep.R
• exprsData_filter.R

Cohort descriptive stats (tableone)

• cohort_stats.R

4. Outlier analysis (PCA)

• pca.R

5. Estimate postmortem degradation (qSVA)

Get expression (coverage) for degradation-associated regions

• run_qsva_degstats.sh

Run qSVA

• qsva.R

Plot

• plot_qsva.R

6. Differential expression analysis

Run edgeR stattests

• edgeR.R

Run edgeR for posthoc tests

Antipsychotic medication in ACC dataset

• posthoc_antipsychotics.R

Other brain regions (PFC and HPC)

• posthoc_brainregions.R

Plot

• plot_edgeR.R

Genelists of differentially expressed genes (DEGs)

• genelists.R

7. Enrichment testing of DEGs

Cell type enrichment analysis (EWCE)

Preprocessing single cell transcriptome datasets

• celldata_Darmanis.R
• celldata_Lake.R

Run EWCE

• ewce_celltypes.R

Plot

• plot_ewce_celltypes.R

Geneset enrichment analysis (ClusterProfiler)

• gsea_kegg.R
• plot_gsea_kegg.R

8. Overlap of DEGs with posthoc tests and other genesets

Annotation references

• anno_geneMap_genomicState.R
• anno_hgnc.R
• anno_rmsk.R
• anno_qsva.R

Updating genesets' gene symbols

Previous analysis of RNA-Seq data

• anno_Zhao_ACC.R
• anno_Darby_HPC.R

SZ-associated genes

• anno_PGC_108loci_Ripke2014.R
• anno_PheGenI.R

Add overlaps to DEG table

• overlaps.R

Plot overlaps

• not finished plot_UpSet.R

9. Correlation/validation with SNCID data

• not finished addl_tests.R