This repository contains scripts used to perform data analyses as detailed in the preprint and final publication by Herrera-Uribe & Wiarda et al., as listed below:
Herrera-Uribe J, Wiarda JE, Sivasankaran SK, Daharsh L, Liu H, Byrne KA, Smith TPL, Lunney JK, Loving CL, Tuggle CK. "Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing". (2021) bioRxiv 2021.04.02.438107. Available at: https://doi.org/10.1101/2021.04.02.438107.
Herrera-Uribe J, Wiarda JE, Sivasankaran SK, Daharsh L, Liu H, Byrne KA, Smith TPL, Lunney JK, Loving CL, Tuggle CK. "Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing". (2021) Frontiers in Genetics. Available at: doi.org/10.3389/fgene.2021.689406.
More detailed information of materials can be found under a heading for each respective directory.
This directory contains scripts used to perform analysis of bulk RNA-seq data from eight sorted cell populations of two porcine PBMC samples. Raw data used for analysis can be found at https://www.ebi.ac.uk/ena/browser/view/PRJEB43826.
Materials in this repository include:
| File Name | File Description |
|---|---|
| PreprocessingMappingAlignmentQualitycontrol | Preprocessing, mapping, alignment, and QC of data |
| EnrichedSpecificGenes | Differential gene expression analysis to determine genes enriched vs specific in sorted cell populations |
Analyses were performed collaboratively by Lance Daharsh & Haibo Liu.
This directory contains scripts used to perform analysis of NanoString data from seven sorted cell populations of two porcine PBMC samples.
Materials in this repository include:
| File Name | File Description |
|---|---|
| Crystal Naostring raw data 21518 KW | Raw data outputs from NanoString assay |
| Crystal Nanostring metadata | Metadata from NanoString assay |
| NanoString Data Analysis | Script used to analyze NanoString data |
Analyses were performed by Haibo Liu.
This directory contains scripts used to perform scRNA-seq analysis from seven porcine PBMC samples.
Selected processed output files/data from analyses described in our scripts can be found in the Ag Data Commons at https://data.nal.usda.gov/dataset/data-reference-transcriptomics-porcine-peripheral-immune-cells-created-through-bulk-and-single-cell-rna-sequencing. Raw data used for analysis can be found at https://www.ebi.ac.uk/ena/browser/view/PRJEB43826.
Materials in this repository include:
| File Name | File Description |
|---|---|
| 001_Background | Background information for initial processing of data |
| 002_Alignment | Alignment of sequencing data to pig genome |
| 003_AmbientRNAcorrection | Estimation and removal of ambient RNA contamination |
| 004_QCfiltering | Removal of poor quality cells and non-expressed genes |
| 005_DoubletRemoval | Removal of highly probable doublets |
| 006_IntegrationAndClustering | Normalization of reads, integration of samples, and clustering of cells |
| 007_ClusterCharacterization | Further characterizing cell clusters to identify general cell types described in previous porcine literature |
| 008_GeneSetEnrichmentAnalysis | Performing gene set enrichment analyses based on gene set signatures for particular sorted cell populations used for bulk RNA-seq analyses; bulk RNA-seq gene sets are compared to single-cell gene profiles to determine enrichment for each individual cell in our scRNA-seq dataset |
| 009_CD4subset | Revamped analysis of only CD4+ T cells from scRNA-seq data |
| 010_GDsubset | Revamped analysis of only CD4+ T cells from scRNA-seq data |
| 011_AgDataCommonsDeposition | Creating data files to deposit on Ag Data Commons |
| RandomForestModeling | Training of a random forest model to cell cluster identities |
| ReferenceBasedLabelTransfer | Cell label prediction and mapping to an external dataset of human PBMCs |
| MitoGenes | List of mitochondrial genes used to calculate percent mitochondrial reads in cells |
| UnfilteredGeneInfo | List of all genes in the v97 genome annotation that were considered in analyses |
Analyses were performed collaboratively by Jayne Wiarda, Sathesh Sivasankaran, and Lance Daharsh. Some scripts for analyses can also be found originally in personal repositories: https://github.com/jwiarda/scRNAseq_PBMCs and https://github.com/satheshsiva/PBMC_scRNAseq.