Data from an experiment
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12 months ago
Chris ▴ 260

Hello all,

I received a single cell data set from a biologist and would appreciate your assistance in understanding the experiment. To minimize costs, the cells from three samples of one condition were combined into a single pool, and a similar approach was taken for the second condition. Each condition consists of 12 fastq files, as depicted in the photo. Within one condition, there are three additional files located in the "filtered_feature_bc_matrix" folder: features.tsv.gz, barcode.tsv.gz, and matrix.mtx.gz.

May I inquire if it would be possible to perform a pseudo-bulk RNA-seq analysis to identify differential gene expression in this case? Is L001-L004 in each condition mean the lane and L001 is a sample? Your guidance and support would be greatly appreciated.

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single-cell • 1.2k views
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L001 to L004 are simply lanes from the FC where the sample ran. Sample files with the same name (but a different L00* number) can be catted together to create a single file. The order of files as you cat these for R1/R2 is critical and should be identical for both.

Sample1_L001_R1.fastq.gz Sample1_L002_R1.fastq.gz  Sample1_L003_R1.fastq.gz  Sample1_L004_R1.fastq.gz  > Sample1.fastq.gz
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Thank you for your previous response. I have a couple of additional questions regarding the experiment.

Regarding the second condition, you see in this photo, is it correct to say that this experiment has a total of 2 samples? Also, what does "FC" mean in this context? Is that flow cell?

Additionally, could you kindly provide guidance on the command I can use to concatenate the 4 fastq files into a single file named "sample1.fastq"? enter image description here

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Looks like there are two samples NS and VV. This data is already analyzed (using cellranger) based on the previous screenshot (where you have the BAM file and cloupe.cloupe file etc). You can look at that analysis using a program called loupe that is provided by 10x Genomics.

FC = Flowcell and in your case Sample1 would be NS and Sample 2 would be VV. I used Sample1 as a placeholder name.

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Based on your previous response, it seems that following the Seurat tutorials may not be necessary in this case. To identify differential gene expression, I should instead use Loupe? Additionally, it appears that pseudo-bulk RNA-seq may not be the most appropriate approach in this scenario.

Is my understanding correct? I appreciate your continued guidance and support! May I know your time zone, PT, CT or ET time?

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Possibly. Take a look at what you received via loupe and make that decision.

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Hi GenoMax, in the first cloupe file (first condition), the browser shows me up and down-regulated genes. So up or down-regulated genes are compared with what level of expression in this case? The second condition also has a cloupe file, so how can I compare two condition to find out which genes is differential expressed genes? Your score is so impressive and I admire your knowledge of bioinformatics!

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Alas my knowledge does not extend to using loupe. Perhaps someone else may chime in.

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I got it. I appreciate your help! Would you answer the first question above that is not related to loupe?

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I've found the 10X tutorial pages extremely helpful. Navigate the mkfastq and count pages as well as relevant pages under understanding outputs, they'll help you understand these files.

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Hi Ram,

I recently read a tutorial on using Loupe and came across the fact that the tutorial used a .cloupe file that combined two conditions into one file. However, I have two separate .cloupe files, each representing a separate condition in my experiment.

My question is how I can identify the differentially expressed genes in this case. Would it be necessary to merge the two .cloupe files into one, or is there another approach I can take?

Thank you for your sharing and assistance.

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I have used the Loupe browser minimally, so I don't think I'll be much help there, but I've found the 10X website really helpful and the 10X support people open to helping too, so you may have better luck asking them specific questions once you read through the documentation.

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Thank you so much! I sent them an email. Have a nice day.

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