Cell Ranger2.0, printed on 12/07/2023
The cellranger pipeline outputs an indexed BAM file containing position-sorted reads aligned to the genome and transcriptome. Reads aligned to the transcriptome across exon junctions in the genome have a large gap in its CIGAR string i.e. 35M225N64M. Each read in this BAM file has Chromium cellular and molecular barcode information attached. Cell Ranger modifies MAPQ values; see the MM tag below. The following assumes basic familiarity with the BAM format. More details on the the SAM/BAM standard are available online.
Chromium cellular and molecular barcode information for each read is stored as TAG fields:
|Z||Chromium cellular barcode sequence that is error-corrected and confirmed against a list of known-good barcode sequences.|
|Z||Chromium cellular barcode sequence as reported by the sequencer.|
|Z||Chromium cellular barcode read quality. Phred scores as reported by sequencer.|
|Z||Chromium molecular barcode sequence that is error-corrected among other molecular barcodes with the same cellular barcode and gene alignment.|
|Z||Chromium molecular barcode sequence as reported by the sequencer.|
|Z||Chromium molecular barcode read quality. Phred scores as reported by sequencer.|
|Z||Sample index read.|
|Z||Sample index read quality. Phred scores as reported by sequencer.|
|Z||Trimmed sequence. For the Single Cell 3' v1 chemistry, this is trailing sequence following the UMI on Read 2. For the Single Cell 3' v2 chemistry, this is trailing sequence following the cell and molecular barcodes on Read 1.|
|Z||Trimmed sequence quality. Phred scores as reported by the sequencer.|
The cell barcode
CB tag includes a suffix with a dash separator followed by a number:
This number denotes what we call a GEM group, and is used to virtualize barcodes in order to achieve a higher effective barcode diversity when combining samples generated from separate GEM chip channel runs. Normally, this number will be "1" across all barcodes when analyzing a sample generated from a single GEM chip channel. It can either be left in place and treated as part of a unique barcode identifier, or explicitly parsed out to leave only the barcode sequence itself.
The following tags will also be present on reads that mapped to the genome and overlapped an exon by at least one base pair. A read may align to multiple transcripts and genes, but it is only considered confidently mapped to the transcriptome it if mapped to a single gene.
|Z||Semicolon-separated list of transcripts that are compatible with this alignment. Transcripts are specified with the |
|Z||Semicolon-separated list of gene IDs that are compatible with this alignment. Gene IDs are specified with the |
|Z||Semicolon-separated list of gene names that are compatible with this alignment. Gene names are specified with |
|i||Set to 1 if the genome-aligner (STAR) originally gave a MAPQ < 255 (it multi-mapped to the genome) and Cell Ranger changed it to 255 because the read overlapped exactly one gene.|
|A||Single character indicating the region type of this alignment (E = exonic, N = intronic, I = intergenic).|