Single Nuclei RNA-Sequencing Analysis Without CellRanger

Question

[16_masques.vole]

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?

Answer 1

[dsull]

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.

[16_masques.vole]

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