10x Genomics
Chromium Single Cell Gene Expression
Cell Ranger5.0, printed on 11/14/2024
Barcoded BAM
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.
BAM Barcode Tags
Chromium cellular and molecular barcode information for each read is stored as TAG fields:
| Tag | Type | Description |
|---|---|---|
CB | Z | Chromium cellular barcode sequence that is error-corrected and confirmed against a list of known-good barcode sequences. |
CR | Z | Chromium cellular barcode sequence as reported by the sequencer. |
CY | Z | Chromium cellular barcode read quality. Phred scores as reported by sequencer. |
UB | Z | Chromium molecular barcode sequence that is error-corrected among other molecular barcodes with the same cellular barcode and gene alignment. |
UR | Z | Chromium molecular barcode sequence as reported by the sequencer. |
UY | Z | Chromium molecular barcode read quality. Phred scores as reported by sequencer. |
TR | 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. |
The cell barcode CB tag includes a suffix with a dash separator followed by a number:
AGAATGGTCTGCAT-1
This number denotes what we call a GEM well, 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.
BAM Alignment Tags
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 if it mapped to a single gene.
| Tag | Type | Description |
|---|---|---|
TX | Z | Present in reads aligned to the same strand as the transcripts in this semicolon-separated list that are compatible with this alignment. Transcripts are specified with the transcript_id key in the reference GTF attribute column. The format of each entry is [transcript_id],[strand][pos],[cigar], where strand is either + or -, pos is the alignment offset in transcript coordinates, and cigar is the CIGAR string in transcript coordinates. |
AN | Z | Same as the TX tag, but for reads that are aligned to the antisense strand of annotated transcripts. |
GX | Z | Semicolon-separated list of gene IDs that are compatible with this alignment. Gene IDs are specified with the gene_id key in the reference GTF attribute column. |
GN | Z | Semicolon-separated list of gene names that are compatible with this alignment. Gene names are specified with gene_name key in the reference GTF attribute column. |
MM | 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. |
RE | A | Single character indicating the region type of this alignment (E = exonic, N = intronic, I = intergenic). |
pa | i | The number of poly-A nucleotides trimmed from the 3' end of read 2. Up to 10% mismatches are permitted. |
ts | i | The number of template switch oligo (TSO) nucleotides trimmed from the 5' end of read 2. Up to 3 mismatches are permitted. The 30-bp TSO sequence is AAGCAGTGGTATCAACGCAGAGTACATGGG. |
xf | i | Extra alignment flags. The bits of this tag are interpreted as follows:
|
Feature Barcode Tags
Sequencing data passed in as a Feature Barcode library type is not aligned to the genome. The BAM file will contain unaligned records for these reads, with the following tags representing the Feature Barcode sequence extracted from the read, and the feature reference it was matched to, if any. The BAM read sequence will contain all the bases outside of the cell barcode and UMI regions.
| Tag | Type | Description |
|---|---|---|
fb | Z | Chromium Feature Barcode sequence that is error-corrected and confirmed against known features barcode sequences from the feature reference. |
fr | Z | Chromium Feature Barcode sequence as reported by the sequencer. |
fq | Z | Chromium Feature Barcode read quality. Phred scores as reported by sequencer. |
fx | Z | Feature identifier matched to this Feature Barcode read. Specified in the id column of the feature reference. |