IRescue 1.0.3

Interspersed Repeats singl-cell quantifier

IRescue - Interspersed Repeats single-cell quantifier

IRescue is a software for quantifying the expression of transposable elements (TEs) subfamilies in single cell RNA sequencing (scRNA-seq) data. The core feature of IRescue is to consider all multiple alignments (i.e. non-primary alignments) of reads/UMIs mapping on multiple TEs in a BAM file, to accurately infer the TE subfamily of origin. IRescue implements a UMI error-correction, deduplication and quantification strategy that includes such alignment events. IRescue's output is compatible with most scRNA-seq analysis toolkits, such as Seurat or Scanpy.

Content

• Installation
  • Using conda
  • Using pip
  • Container (Docker/Singularity)
• Usage
  • Quick start
  • Output files
  • Load IRescue data with Seurat
• Cite

Installation

Using conda (recommended)

We recommend using conda, as it will install all the required packages along IRescue.

conda create -n irescue -c conda-forge -c bioconda irescue

Using pip

If for any reason it's not possible or desiderable to use conda, it can be installed with pip and the following requirements must be installed manually: python>=3.7, samtools>=1.12 and bedtools>=2.30.0.

pip install irescue

Container (Docker/Singularity)

Docker and Singularity containers are available for each conda release of IRescue. Choose the TAG corresponding to the desired IRescue version from the Biocontainers repository and pull or execute the container with Docker or Singularity:

# Get latest biocontainers tag (with curl and python3, otherwise check the above link for the desired version/tag)
TAG=$(curl -s -X GET https://quay.io/api/v1/repository/biocontainers/irescue/tag/ | python3 -c 'import json,sys;obj=json.load(sys.stdin);print(obj["tags"][0]["name"])')

# Run with Docker
docker run quay.io/biocontainers/irescue:$TAG irescue --help

# Run with Singularity
singularity exec https://depot.galaxyproject.org/singularity/irescue:$TAG irescue --help

Usage

Quick start

The only required input is a BAM file annotated with cell barcode and UMI sequences as tags (by default, CB tag for cell barcode and UR tag for UMI; override with --CBtag and --UMItag). You can obtain it by aligning your reads using STARsolo.

RepeatMasker annotation will be automatically downloaded for the chosen genome assembly (e.g. -g hg38), or provide your own annotation in bed format (e.g. -r TE.bed).

irescue -b genome_alignments.bam -g hg38

If you already obtained gene-level counts (using STARsolo, Cell Ranger, Alevin, Kallisto or other tools), it is advised to provide the whitelisted cell barcodes list as a text file, e.g.: -w barcodes.tsv. This will significantly improve performance.

IRescue performs best using at least 4 threads, e.g.: -p 8.

Output files

IRescue generates TE counts in a sparse matrix format, readable by Seurat or Scanpy:

IRescue_out/
├── barcodes.tsv.gz
├── features.tsv.gz
└── matrix.mtx.gz

Load IRescue data with Seurat

To integrate TE counts into an existing Seurat object containing gene expression data, they can be added as an additional assay:

# import TE counts from IRescue output directory
te.data <- Seurat::Read10X('./IRescue_out/', gene.column = 1, cell.column = 1)

# create Seurat assay from TE counts
te.assay <- Seurat::CreateAssayObject(te.data)

# subset the assay by the cells already present in the Seurat object (in case it has been filtered)
te.assay <- subset(te.assay, colnames(te.assay)[which(colnames(te.assay) %in% colnames(seurat_object))])

# add the assay in the Seurat object
seurat_object[['TE']] <- irescue.assay

The result will be something like this:

An object of class Seurat 
32276 features across 42513 samples within 2 assays 
Active assay: RNA (31078 features, 0 variable features)
 1 other assay present: TE

Cite

Polimeni B, Marasca F, Ranzani V, Bodega B. IRescue: single cell uncertainty-aware quantification of transposable elements expression. bioRxiv 2022.09.16.508229; doi: https://doi.org/10.1101/2022.09.16.508229