Hi! We have recently performed Chromium Next GEM single cell 3' version 3.1 (Dual Index), where we have used four different hashtag barcodes (TotalseqB) in each run (each run contains four time points) and performed four runs in total. We have also used ten antibodies in the panel to measure the expression of certain proteins.
Now I have received 8 sets of fastq files (four from the antibody libraries and four from the cell surface protein libraries, each set containing one R1 and one R2 fastq files) from the sequencing facilities. Now I want to demultiplex these fastq files so that I get fastq files for each timepoints.
I have tried with cellranger multi according to (https://kb.10xgenomics.com/hc/en-us/articles/4407386498957-I-used-antibody-tags-for-cell-surface-protein-capture-and-cell-hashing-with-Single-Cell-3-chemistry-How-can-I-use-Cell-Ranger-to-analyze-my-data-?source=search), but it does not work as I can't edit the copied antibody fastq files as suggested here (files are too big to be opened by a text editor).
Can anyone please suggest me any tutorial or tools for demultiplexing and preprocessing these data, so that I can later use it for Seurat? Thanks in advance!
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s.sajib63 •
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You do not need to open fastq files in texteditor to run CellRanger (which indeed is close to impossible). All you have to do is to specify the barcode sequence in the reference CSV file. What part of the manual makes you think that you have to modify the fastq files itself?
Edit: Ah ok, I misread your question, my bad.
Hi! Thanks for the reply. I was talking about the part which says "However, given that you used the same antibody library for dual purposes (cell surface protein capturing + cell hashing), you will need to make a copy of your antibody FASTQ files and also make a tiny edit (for example, delete the first read) in the copy. Please make sure you delete the same read in R1 and R2 reads FASTQs (I1 and I2 as well, if available)." in the link mentioned above. So I don't have to edit the copy fastq file?
The directions indeed say that you need to remove at least one read from each file (that is how I read the directions). You will likely need to uncompress the files on the fly and use
sedto remove the first 4 lines and then recompress the data. While the edit istinyit is not simple.You could
bgzip -d your_file.fq.gz. Then remove the first 4 lines.sed -i -e '1,4d' your_file.fq. And then recompress edited filebgzip your_file.fq.Hi Sajib,
I am also trying to demultiplex my fastq files. Did you figure out how to delete a read from fastq file. Any help would be much appreciated. TIA.