BAM (Binary Alignment Map) files are a binary format used to store sequencing data aligned to a reference genome.

The cellranger count pipeline generates an indexed BAM file named possorted_genome_bam.bam. This file contains position-sorted reads aligned to the genome and transcriptome, along with unassigned reads. If Feature Barcode libraries are included in the analysis, the BAM file will also include both aligned and unaligned records for each library type (e.g., Antibody Capture, CRISPR Guide Capture, 3' Cell Multiplexing, Antigen Capture).

The cellranger multi pipeline produces two BAM files. It separates the reads into unassigned reads, which are stored in unassigned_alignments.bam, and reads that are assigned to samples, which are stored in sample_alignments.bam.

BAM files can be used for troubleshooting reads that were unassigned or for converting BAM files back to FASTQ files. Each read in this BAM file has Chromium cellular and molecular barcode information attached. The following tables assume basic familiarity with the BAM format. The Type column refers to the type of value stored in the tag (see the SAM/BAM standard) documentation for details.

Chromium cellular and molecular barcode information for each read is stored in the following TAG fields:

The cell barcode CB tag includes a suffix with a dash separator followed by a number:

Copy
AAACCCAAGGAGAGTA-1

This number denotes the GEM well, and is used to virtualize barcodes in order to achieve a higher effective barcode diversity when combining samples generated from separate GEM well channel runs. Normally, this number will be "1" across all barcodes when analyzing a sample generated from a single GEM well 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. Reads aligned to the transcriptome across exon junctions in the genome will have a large gap in their CIGAR string i.e., 35M225N64M. A read may align to multiple transcripts and genes, but it is only considered confidently mapped to the transcriptome if it is mapped to a single gene (see this page for methods to check for multi-mapped reads). Cell Ranger modifies MAPQ values; see the mm tag below.

Some aspects of the BAM file are particular to Fixed RNA Profiling samples. The mapping quality (MAPQ) values described here do not apply to 3' or 5' Gene Expression data.

A read that multi-maps to the reference transcriptome, but maps uniquely to a single probe, is considered confidently mapped to that probe and its gene. The mapping quality has the following definition:

The following tags represent the Feature Barcode sequence extracted from the read and the feature reference it was matched to, if any. Sequencing reads passed in as a Feature Barcode library type are not aligned to the genome and the BAM file will contain unassigned records for these reads. See the Feature Barcode Extraction Pattern section for more details about the alignment algorithm. The BAM read sequence will contain all the bases outside of the cell barcode and UMI regions.

This Analysis Guide tutorial walks users through the process of identifying records in the BAM file that contribute to UMI counting: Navigating 10x Genomics Barcoded BAM Files. Note: 10x Genomics does not provide support for community-developed tools and makes no guarantees regarding their function or performance. Please contact tool developers with any questions. If you have feedback about Analysis Guides, please email [email protected].