HorvathLab / NGS

Next-Gen Sequencing tools from the Horvath Lab
https://horvathlab.github.io/NGS/
MIT License
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Clarification on Workflow for SCReadCounts Application #20

Open Giovanna0806 opened 6 months ago

Giovanna0806 commented 6 months ago

Hello Nathan again, I am currently using your method described in the paper "SCReadCounts: estimation of cell-level SNVs expression from scRNA-seq data" for cell-specific allele expression analysis. I would appreciate your clarification on the proper workflow.

Based on the paper, it appears that the analysis can be conducted on the raw pooled sequencing data. Here is my understanding of the correct workflow:

  1. Align the raw pooled sequencing data.
  2. Call variants from the pooled alignments using GATK.
  3. Filter the variant calls, retaining high-quality biallelic positions supported by a minimum of 50 sequencing reads for both the variant and reference alleles.
  4. Provide the filtered variant lists to SCReadCounts, along with the corresponding alignments and the STARsolo generated list of error-corrected barcodes.

Is this the correct approach, or is it necessary to split the raw pooled sequencing data into single cells at an earlier stage? If splitting into single cells is required, could you please provide the correct workflow for this process, as well as any scripts or specific instructions for using SCReadCounts in this context?

Thank you very much for your time and assistance. Giovanna