Entering edit mode
3.0 years ago
david.f.stein ▴ 10
I am new to scRNA-seq analysis. I have used CellRanger's count pipeline to get gene expression matrices for downstream analysis with Seurat. I am attempting to get a similar expression matrix from Fluidigm C1 data to incorporate into my Seurat analysis, but I'm not sure what the best software to use is. On the Fluidigm website they offer a Singular Analysis Toolset but mention that it is not appropriate for bulk gene expression analysis. I have a couple hundred GB's of data and need to analyze them on my cluster. Does anyone know what software I should use?
Hey, can you help us a bit further by stating exactly the files that you currently have (from Fluidigm), and also stating the exact experiment that you conducted (library prep)? From what I gather, your Fluidigm data is from bulk RNA-seq, so, Seurat would not even be the natural choice for it.
Sorry, I didn't know what information was relevant to include. I have paired fastq files. Also this is not my experiment, its data from SRA. I am using Seurat because I have data from multiple species and they have a workflow for multispecies and multiplatform data integration. If theres a more appropriate tool for this task though I'm open to it. The other two datasets I had came from 10x as I said. I see that cellranger uses STAR to do their counting. Maybe I should use that for the Fluidigm data as well?
Slide number 4 shows a potential workflow you can use for your fluidigm data.
Thanks! Kallisto seems like a reasonable option. But would it be better to use STAR considering the other datasets I'm incorporating were counted with CellRanger count which uses STAR?
You can. I am not sure if the data you have has already been separated into cells? I don't have first hand experience with fluidigm data (disclaimer).
Hmm, is there a way to figure out if they have been separated. This is the relevant preperation description from the authors "Around 40,000 cells were sorted for each single-cell experiment into 300 μl of 0.8× PBS, and at least two microfluidic C1 chips (Fluidigm, 96 capture sites) were used per experiment. For stage 40 and stage 44 limb bud experiments, dissociated cells were directly used for single-cell isolation on microfluidic chips without FACS enrichment".
I tried to see if there were any resources available but have not found any. What does your data look like? Are there as many fastq files as there are samples?