Analysis of sc-RNA data

Question

[halo22]

Hello All,

I am trying to analyze data for a single cell RNA sequencing experiment, for QC and normalization I am considering using the scater package. There are a few things I would like to know before starting analyzing this dataset. All your help and suggestions are much appreciated. This is my first attempt to analyze sc-RNA, I apologize in advance if my questions are confusing. Questions: 1) The sequencing lab is using an unpublished protocol, they have provided read counts and spike-ins file separately. Do I need to combine these two files? I am considering this, for the "feature_controls" option for calculateQCMetrics method.

2) After doing the initial QC, I see that total counts for all my wells(cells) is almost >50k+ and the number of genes detected is above 10k. I am removing genes that have 0 expression, I am also filtering genes with very low average nonzero expression across all cells(using a mean of counts across all cells). Do I need to do any other filtering for both cells and genes?

Also, I would greatly appreciate, if you could direct me to a sample reference analysis.

Answer 1

[Mike]

Hope this link will help you...

Single Cell RNAseq data analysis protocol

Answer 2

[esnolli]

Hello halo22,

I am new here, as well as in the world in Bioinformatics. However, I am trying to analyze scRNA-seq data as you may be doing at this moment.

For scater there is a wonderful vignette (https://bioconductor.org/packages/devel/bioc/vignettes/scater/inst/doc/vignette.html) and I found also this paper with a "quick" protcol for analyzing scRNA-seq data (https://www.bioconductor.org/help/workflows/simpleSingleCell/).

Unfortunetaly, I think no standard protocol for scRNA seq is available, neither for bulk RNA seq or other studies. You must consider the specificities of your study and try to always adapt to your data with the proper tools.

Hope to have helped you.

Cheers,

Esteve