Overview

This repository provides initial analysis of K562 and RPE1 Perturb-seq data after reprocessing with the scUTRquant pipeline to resolve 3'UTR isoforms rather than gene counts. It includes both some preprocessing code used to created the metadata tables used in the scUTRquant pipeline, and the initial stages of filtering, summarization to pseudobulk, clustering of the KD6 (K562 6-day essential screen) data, differential gene and isoform expression testing, and a replication analysis using the RD7 (RPE1 7-day essential screen) data.

Additionally analyses downstream of this are included in the scUTRquant-figures repository.

A preprint of the results are reported in Fansler et al., bioRxiv, 2023.

Organization

The folders in the repository have the following purposes:

• analysis - primary source code and rendered HTMLs of R Markdown
• envs - Conda environment YAML files for recreating the execution environment
• img - output images
• metadata - output tables used in pipeline
• scripts - additional scripts for data format conversions
• tbl - output result tables

All code is expected to be executed with this repository as the present working directory. If opening as an R Project in RStudio, make sure to set the Project folder as the working directory.

Source Code

The primary source code is found in the analysis folder. Files are numbered in the original order of execution, though the order does not imply strict necessity (most analyses here can be independently executed).

The analysis/preprocessing/*_scutrquant_inputs.Rmd files were used to populate the metadata tables used by the scUTRquant pipeline. These should not be necessary to rerun if starting scUTRquant outputs.

Execution Environments

The R instances used to execute the files was captured both in the rendered RMDs themselves (see Runtime Details section in HTMLs) and provided as YAML files in the envs folder.

To recreate on arbitrary platforms (Linux or MacOS), we recommend using Micromamba and the minimal YAML (*.min.yaml):

micromamba create -n bioc_3_16 -f envs/bioc_3_16.min.yaml
micromamba activate bioc_3_16

A fully-solved environment capture is also provided (*.full.yaml). This is only expected to recreate on the osx-64 platform and is primarly intended for exact replication and a statement of record.