Why did I get very poor scRNAseq files through STARSolo, Galaxy.
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6 months ago
Youyy • 0

I have raw data from 10x single cell RNA seq v3. Both R1 and R2 are fastq.gz files, each has a size about 20G.

I was running STARSolo by following the https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/scrna-preprocessing-tenx/tutorial.html

“Input Type”: Paired collection of barcode and cDNA reads param-file  
“Collection of Pairs”: the name of your paired collection param-file  
“RNA-Seq Cell Barcode Whitelist”: 3M-february-2018.txt.gz 
“Custom or built-in reference genome”: Use a built-in index 
“Reference genome with or without an annotation”: use genome reference without builtin gene-model 
“Select reference genome”: Human (Homo Sapiens): hg19 Full  
“Gene model (gff3,gtf) file for splice junctions”: Homo_sapiens.GRCh37.75.gtf In 
“Advanced Settings”: 
“Configure Chemistry Options”: Cell Ranger v3

l I got the three files, genes, barcodes and matrix. And then I was trying to follow the Seurat instruction on R as follows:

https://satijalab.org/seurat/v3.2/pbmc3k_tutorial.html

But however, in Seurat, I only got 43 cells and was unable to run dimentional reduction RunPCA(), pbmc <- RunPCA(pbmc, features = VariableFeatures(object = pbmc)) because the default number of npcs(number of PCs) is 50, then I reduced to 14 15 40 and tried to cluster the cells, however, I only got one cluster. I am wondering if anyone has the same problem with me? Thank you so much.

note: I didn't pair my R1 and R2 files, I uploaded them separately, I think this is a case. I was re-running it with the paired file(paired R1 and R2 raw data), now I got much more larger output than the first time, I think this time will be better, I will see it. The problem has been solved.

Galaxy STARSolo R Seurat scRNA-seq • 410 views
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I appreciate if anyone can figure out any problem.

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Without code it is almost impossible to help. Getting only 43 cells suggests that something is very wrong in your preprocessing, or that library quality is abysmal. You will need to add code and details for further help.

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Thank you. I am not sure how to extract the code from Galaxy. Last time I didn't pair the R1 and R2 files, but I did this time, it's still running.

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That may have been one of the problems. Are you simply following the tutorial i.e. not using your own data? If that is the case stick with directions in the tutorial.

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