Need to specify "--chemistry" argument in "cellranger count"

Question

[Aaron]

I'm using CellRanger to analyze some RNA-seq data I have but I'm having a bit of trouble knowing if I'm doing the preprocessing steps correctly. This is the folder of Fastqs that I have:

Control_GEX_S1_L003_I1_001.fastq.gz    Treatment_BCR_S6_L004_R2_001.fastq.gz
Control_GEX_S1_L003_I2_001.fastq.gz    Treatment_GEX_S2_L003_I1_001.fastq.gz
Control_GEX_S1_L003_R1_001.fastq.gz    Treatment_GEX_S2_L003_I2_001.fastq.gz
Control_GEX_S1_L003_R2_001.fastq.gz    Treatment_GEX_S2_L003_R1_001.fastq.gz
Control_GEX_S1_L004_I1_001.fastq.gz    Treatment_GEX_S2_L003_R2_001.fastq.gz
Control_GEX_S1_L004_I2_001.fastq.gz    Treatment_GEX_S2_L004_I1_001.fastq.gz
Control_GEX_S1_L004_R1_001.fastq.gz    Treatment_GEX_S2_L004_I2_001.fastq.gz
Control_GEX_S1_L004_R2_001.fastq.gz    Treatment_GEX_S2_L004_R1_001.fastq.gz
FASTQ Files-selected (3).zip           Treatment_GEX_S2_L004_R2_001.fastq.gz
FASTQ Files-selected (4).zip           Treatment_TCR_S4_L003_I1_001.fastq.gz
FASTQ Files-selected.zip               Treatment_TCR_S4_L003_I2_001.fastq.gz
Matrix Files (1).zip                   Treatment_TCR_S4_L003_R1_001.fastq.gz
Treatment_BCR_S6_L003_I2_001.fastq.gz  Treatment_TCR_S4_L003_R2_001.fastq.gz
Treatment_BCR_S6_L003_R1_001.fastq.gz  Treatment_TCR_S4_L004_I1_001.fastq.gz
Treatment_BCR_S6_L003_R2_001.fastq.gz  Treatment_TCR_S4_L004_I2_001.fastq.gz
Treatment_BCR_S6_L004_I1_001.fastq.gz  Treatment_TCR_S4_L004_R1_001.fastq.gz
Treatment_BCR_S6_L004_I2_001.fastq.gz  Treatment_TCR_S4_L004_R2_001.fastq.gz
Treatment_BCR_S6_L004_R1_001.fastq.gz

As I understand it, there are four samples here: "Control_GEX," "Treatment_GEX," "Treatment_TCR," and "Treatment_BCR" (although I wasn't sure if there may be at least 6 samples overall because I see that there are "S1," "S2," "S4," "S6" after the sample names). I've been running "cellranger count" on each sample. Is this generally correct? It runs successfully for the first three samples but it generates this error for "Treatment_BCR":

[error] Pipestance failed. Error log at:
Treatment_BCR/SC_RNA_COUNTER_CS/SC_MULTI_CORE/MULTI_CHEMISTRY_DETECTOR/_GEM_WELL_CHEMISTRY_DETECTOR/DETECT_COUNT_CHEMISTRY/fork0/chnk0-ufe99c8e697/_errors

Log message:
Unable to distinguish between [SC5P-R2, SC3Pv2] chemistries based on the R2 read mapping for Sample Treatment_BCR in "/projects/p32525/RNAseq_data/Fastqs".
Total Reads          = 100000
Mapped reads         = 72021
Sense reads          = 13294
Antisense reads      = 26340

In order to distinguish between the 3' vs 5' assay configuration the following conditions need to be satisfied:
- A minimum of 1000 confidently mapped reads
- A minimum of 5.0% of the total reads considered needs to be confidently mapped
- The number of sense reads need to be at least 2x compared to the antisense reads or vice versa

Please validate the inputs and/or specify the chemistry via the --chemistry argument.


Waiting 6 seconds for UI to do final refresh.
Pipestance failed. Use --noexit option to keep UI running after failure.

2021-12-26 16:03:47 Shutting down.

I'm not quite sure how to resolve this - "cellranger count" worked for all the other samples, and I'm not quite sure what "--chemistry" argument I would specify here. I'm working with data someone else generated and I don't have access to the experimental set up - would I find or be able to infer that "--chemistry" argument?

EDIT:

Also, I haven't been able to find a clear explanation of what "GEX" means, and why "BCR," "TCR," and "GEX" are used - is this multiplexing? Also I'm not quite sure in a very basic way how "BCR," "TCR," and "GEX" are used

[GenoMax]

So this single cell immune data: https://www.10xgenomics.com/products/single-cell-immune-receptor-mapping and https://www.10xgenomics.com/products/single-cell-immune-profiling

You can find tutorials/software and all relevant information here: https://support.10xgenomics.com/single-cell-vdj and https://support.10xgenomics.com/single-cell-vdj/software/overview/welcome

[jv]

GEX = gene expression
TCR = T-cell receptor
BCR = B-cell receptor

Answer 1

[kmaroney]

Hi, I believe you need to follow this tutorial: https://www.10xgenomics.com/resources/analysis-guides/demultiplexing-and-analyzing-5%E2%80%99-immune-profiling-libraries-pooled-with-hashtags

It is NOT straightforward, but essentially you are going to need to separate your fastqs out into individual folders first, make a config file for each individual in a folder called "final_analysis" specifying GEX, TCR, and BCR, and then also use the --chemistry 5' (I think) argument to specifiy 5' or 3' depending on the kit. You'd have to do this twice if you hashtag samples (the features are simply separate hashtags, then the second time the features are antibody-oligos for markers)

I'm not sure why you get that message though, as I had an experiment where one of my demultiplexed samples only returned ~46 cells (bad sample prep, low number of cells sorted) whereas the others still had < 200 cells, but both your cell number and number mapped reads seem fine.

As for the naming, "TCR" = your TCR library (pulled from the raw reads returned from your sequencing amplification, possibly also run with other indices - each 10X library (GEX, TCR, BCR, hashtag) is prepared separately on the GEM well, resulting in an individual index which is amplified as a library of cDNA -> you get a fasta/fastq file back for this, both R1 and R2, one of which has the feature barcodes (which should include all the UMI's/whatever is called for hashing or genes. Still a little foggy on this, but I've at least gotten it to work. I think that link should be enough to at least get started. I could share some code for what I've been working on which may be easier to follow because this is much more complicated than I believe it needs to be :/