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Single Nuclei RNA-Sequencing Analysis Without CellRanger

I am attempting a multiome single nuclei RNA-Sequencing analysis. I have gene expression and ATAC data. The data was generated using 10X genomics pipeline. The mapping from Cellrangers-arc count function is woefully inadequate and I want to manually map the single nuclei reads and generate counts (and other data that cellranger outputs in the h5 file) from that BAM file. I am stuck however, because I cannot find a way of incorporating the 10X barcodes into my BAM file. In the BAM file generated by cellranger-arc count, there are CB:Z:__barcode__ tags assigned to each alignment. I cannot find a way of achieving this without using cellranger. Does anyone have any advice?

cellranger single-cell snrna-seq
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written by 16_masques.vole • 16_masques.vole

1 answer

Hmm, what aligner are you using? STARsolo will automatically put the barcodes into the CB:Z: tag in the BAM file.

Otherwise, I believe you'll have to do it manually. You can do your alignment (specifying you want the barcodes soft-clipped) and then go into your aligned BAM file, extract the barcode and put it into the CB:Z: column. Or, you can manually go through your FASTQ file, match the read names to that in your BAM file, and manually extract the barcode + edit your BAM file. You can edit your BAM file by writing some scripts of your own and using samtools/pysam/etc.

Anyway, those are some of my suggestions.

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written by dsull dsull

Thanks. I am using HiSAT2 as my aligner but I will checkout STARsolo

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written by 16_masques.vole • 16_masques.vole

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