GEO datasets-Single cell RNAseq analysis using seurat

Question

[LionisOne]

I'm trying to look through a published dataset to check for gene expression for my own project. I am having trouble finding materials to teach me how to analyze single cell RNA sequencing data on Seurat R from the GEO NCBI database. Since multiple barcodes.tsv.gz,genes.tsv.gz and matrix.mtx.gz files are there in this datasets. How to analyze those? Your help is much appreciated. Thanks in advance !

Dataset

GSE128423, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE128423

datasets looks like this

GSM3674224_std1.barcodes.tsv.gz     GSM3674231_ctrl_16May.matrix.mtx.gz GSM3674239_b1.genes.tsv.gz
GSM3674224_std1.genes.tsv.gz        GSM3674232_ctrl_26May.barcodes.tsv.gz   GSM3674239_b1.matrix.mtx.gz
GSM3674224_std1.matrix.mtx.gz       GSM3674232_ctrl_26May.genes.tsv.gz  GSM3674240_b2.barcodes.tsv.gz
GSM3674225_std2.barcodes.tsv.gz     GSM3674232_ctrl_26May.matrix.mtx.gz GSM3674240_b2.genes.tsv.gz
GSM3674225_std2.genes.tsv.gz        GSM3674233_ctrl_7Jun.barcodes.tsv.gz    GSM3674240_b2.matrix.mtx.gz
GSM3674225_std2.matrix.mtx.gz       GSM3674233_ctrl_7Jun.genes.tsv.gz   GSM3674241_b3.barcodes.tsv.gz
GSM3674226_std3.barcodes.tsv.gz     GSM3674233_ctrl_7Jun.matrix.mtx.gz  GSM3674241_b3.genes.tsv.gz
GSM3674226_std3.genes.tsv.gz        GSM3674234_ctrl_8May.barcodes.tsv.gz    GSM3674241_b3.matrix.mtx.gz
GSM3674226_std3.matrix.mtx.gz       GSM3674234_ctrl_8May.genes.tsv.gz   GSM3674242_b4.barcodes.tsv.gz

[GenoMax]

This is standard output of cellranger count analysis. You can use these files with seurat following one of the tutorials below.

https://www.fimm.fi/sites/default/files/Seurat-guideline-10x.pdf
https://hbctraining.github.io/scRNA-seq/lessons/03_SC_quality_control-setup.html

[LionisOne]

@GenoMax,

Thank you very much for your reply. I know how to use Seurat from Cellranger output. But I'm not sure how to deal with multiple files (several barcodes.tsv.gz, several matrix files, etc.)

Answer 1

[predeus]

If you place three files dedicated to each sample (barcodes, genes, and matrix) into a folder, you can read it into a sparse matrix object using Read10x function from Seurat. This matrix can then be converted into a Seurat object using CreateSeuratObject.

Tricky part is figuring out if you want to just "stitch" the samples, or actually properly integrate them. If these are replicates of the same sample, you need to check for overlapping barcodes, but otherwise you can simply merge the obtained Seurat objects. If they are different biological samples, do proper integration, like described in Seurat vignette.

[mogollon.garcia]

Hi! Predeus. I'm working with the dataset GSE121861, but its don't have a barcode file. I can run this data in Seurat without this file?. Thanks for the comments.

[predeus]

it's a 10x experiment, so it should have the barcodes. They have deposited it in some weird format though. You should be able to load the provided supplementary files (csv.gz files on the bottom of this page: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE121861) as an expression matrix, and then create Seurat object from the matrix with CreateSeuratObject

Otherwise, you can always download the SRA files and process the data from the scratch using STARsolo or Cell Ranger..

[LionisOne]

@Predeus, Thank you very much!

[Pierre Lindenbaum]

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