Single Cell Rna Seq Using BD Rhapsody

Question

[abbas.waseem.gcu]

Hello Everyone. I am working with scRNA for the first time. I have paired end raw read sequenced through BD Rhapsody. Please suggest me a pipeline for using this from quality control to single cell cluster analysis. Any workflow suggested for this for this from Galaxy Europe Tools.

Answer 1

[GenoMax]

See answer here: BD Rhapsody software - single cell seq

I don't think you can do this on Galaxy.

[dsull]

I think a good solution would be kallisto/bustools (or other solutions that work, like STARsolo or alevin-fry).

E.g. For kallisto/bustools, pip install kb_python, build your kallisto index with the kb ref command, and then do the quantification with kb count -x BDWTA.

(BDWTA tells kallisto to parse the reads based off BD Rhapsody structure).

You then get count matrices which you can load into scanpy or seurat for downstream analysis and QC'ing like you would with 10X data.

[GenoMax]

Good to know. I was unaware that kb can understand BD data. I was under the impression that one needed to use BD software for the initial processing since they use the identical indexes for two things. Or are you referring to data that has been demultiplexed by BD software.

[dsull]

I'm referring to non-multiplexed data where the only thing you need to deal with are the three 9-bp cell barcodes separated by linkers. I think that's all you need for the WTA kit.

For the multiplexing or targeting or multiomics BD kits, you'd need to do the demultiplexing yourself or with BD software (you need to ask BD for the specific sequences though).

Answer 2

[eduardo.andres.leon]

I'm using Seurat. The protocol is quite similar to other scRNASeq and Seurat has a really good documentation. In my expiricence most of the filtering stepts are similar to 10X with the exception of mt-RNA. Data from Rhapsody has fewer mt-RNA that 10x