The spaceranger count pipeline outputs metrics_summary.csv which contains a number of key metrics about the barcoding and sequencing process.
| Metric | Description |
|---|---|
Number of Spots Under Tissue | The number of barcodes associated with a spot under tissue. |
Number of Reads | Total number of read pairs that were assigned to this library in demultiplexing. |
Mean Reads per Spot | The number of reads, both under and outside of tissue, divided by the number of barcodes associated with a spot under tissue. |
Mean Reads Under Tissue per Spot | The number of reads under tissue divided by the number of barcodes associated with a spot under tissue. |
Fraction of Spots Under Tissue | The fraction of the spots under the tissue. |
Median Genes per Spot | The median number of genes detected per spot under tissue-associated barcode. Detection is defined as the presence of at least one UMI count. |
Median UMI Counts per Spot | The median number of UMI counts per tissue covered spot. |
Valid Barcodes | Fraction of reads with barcodes that match the whitelist after barcode correction. |
Valid UMIs | Fraction of reads with valid UMIs; i.e. UMI sequences that do not contain Ns and that are not homopolymers. |
Sequencing Saturation | The fraction of reads originating from an already-observed UMI. This is a function of library complexity and sequencing depth. More specifically, this is the fraction of confidently mapped, valid spot-barcode, valid UMI reads that had a non-unique (spot-barcode, UMI, gene). |
Q30 Bases in Barcode | Fraction of spot barcode bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q lesser than or equal to 2) bases from the denominator. |
Q30 Bases in RNA Read | Fraction of RNA read bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q lesser than or equal to 2) bases from the denominator. This is Read 2 for the Visium v1 chemistry. |
Q30 Bases in UMI | Fraction of UMI bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q lesser than or equal to 2) bases from the denominator. |
Reads Mapped to Genome | Fraction of reads that mapped to the genome. |
Reads Mapped Confidently to Genome | Fraction of reads that mapped uniquely to the genome. If a gene mapped to exonic loci from a single gene and also to non-exonic loci, it is considered uniquely mapped to one of the exonic loci. |
Reads Mapped Confidently to Intergenic Regions | Fraction of reads that mapped uniquely to an intergenic region of the genome. |
Reads Mapped Confidently to Intronic Regions | Fraction of reads that mapped uniquely to an intronic region of the genome. |
Reads Mapped Confidently to Exonic Regions | Fraction of reads that mapped uniquely to an exonic region of the genome. |
Reads Mapped Confidently to Transcriptome | Fraction of reads that mapped to a unique gene in the transcriptome. The read must be consistent with annotated splice junctions. These reads are considered for UMI counting. |
Reads Mapped Antisense to Gene | Fraction of reads confidently mapped to the transcriptome, but on the opposite strand of their annotated gene. A read is counted as antisense if it has any alignments that are consistent with an exon of a transcript but antisense to it, and has no sense alignments. |
Fraction Reads in Spots Under Tissue | The fraction of valid-barcode, confidently-mapped-to-transcriptome reads with spot-associated barcodes. |
Total Genes Detected | The number of genes with at least one UMI count in any tissue covered spot. |
The spaceranger count for FFPE pipeline outputs metrics_summary.csv which differs from spaceranger count with regard to a few key metrics.
| Metric | Description |
|---|---|
Reads Mapped to the Probe Set | Fraction of reads that map with at least one read half to the probe reference. |
Reads Mapped Confidently to the Probe Set | Fraction of reads that map uniquely with both read halves to the probe reference. |
Reads Mapped Confidently to the Filtered Probe Set | Fraction of reads that map uniquely with both read halves to the filtered probe reference. The probe reference is filtered to remove genes/features where one or more of the probes targeting this feature might hybridize and ligate at a non targeted loci. This metric will be "None" when probe filtering is disabled. |
Genes Detected | The number of unique genes from the filtered probe set with at least one UMI count in any tissue covered spot. |
Number of Genes | The number of genes as defined by the probe set. |
Number of Genes greater than or equal to 10 UMIs | Number of genes with at least 10 filtered UMIs from tissue-associated barcodes. These genes are used to calculate per-gene enrichments. |
Space Ranger computes sequencing quality and application results metrics on each supported library, which currently are Gene Expression and Antibody Capture. These metrics will be computed and displayed only when one of these library types was used. This page describes Antibody Capture libraries metrics for QC of the library preparation and sequencing of Protein Expression libraries, which appear in the metrics_summary.csv file and on the web_summary.html page.
| Metric | Description |
|---|---|
Antibody Number of Reads | Total number of reads. |
Mean Antibody Reads per Spot | The number of reads, both under and outside of tissue, divided by the number of barcodes associated with a spot under tissue. |
Valid Antibody Barcodes | Fraction of reads with a spot-barcode found in or corrected to one that is found in the whitelist. |
Valid Antibody UMIs | Fraction of reads with valid UMIs; i.e. UMI sequences that do not contain Ns and that are not homopolymers. |
Antibody Sequencing Saturation | Fraction of antibody library reads originating from an already-observed UMI. This is a function of library complexity and sequencing depth. More specifically, this is a ratio where: the denominator is the number of reads with a recognized antibody barcode, valid cell-barcode, and valid UMI, and the numerator is the subset of those reads that had a non-unique combination of (spot-barcode, UMI, antibody barcode). |
Q30 Bases in Antibody Barcode | Fraction of cell barcode bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q lesser than or equal to 2) bases from the denominator. |
Q30 Bases in Antibody Read | Fraction of bases from the read containing the antibody barcode with Q-score greater than or equal to 30, excluding very low quality/no-call (Q lesser than or equal to 2) bases from the denominator. This is Read 2 for the Visium v2 chemistry. |
Q30 Bases in Antibody UMI | Fraction of UMI bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q lesser than or equal to 2) bases from the denominator. |
Fraction Antibody Reads | Fraction of reads that contain a recognized antibody barcode |
Fraction Antibody Reads Usable | Fraction of reads that contain a recognized antibody barcode, a valid UMI, and a spot-associated barcode. |
Antibody Reads Usable per Spot | Number of antibody reads usable divided by the number of spot-associated barcodes. |
Fraction Unrecognized Antibody | Among all reads, the fraction with an unrecognizable antibody barcode |
Antibody Reads in Spots Under Tissue | Among Antibody library reads with a recognized antibody barcode, a valid UMI, and a valid barcode, the fraction associated with a spot under tissue. |
The spaceranger aggr pipeline outputs summary.json that contains metrics relating to the aggregated datasets. Note: Square brackets denote a variable that depends on the pipeline input, such as, [library_id]_frac_reads_kept means that if your aggregation contains two libraries with IDs sample123 and sample456, there are two output metrics sample123_frac_reads_kept and sample456_frac_reads_kept.
For aggregated datasets that contain both Gene and Protein Expression libraries, there are additional metrics for Protein Capture that include [Antibody] in the metric name.
| Metric | Description |
|---|---|
filtered_bcs_transcriptome_union | The estimated number of barcodes associated with a spot under tissue, summed across all input libraries. |
[pre/post]_total_reads | Total number of sequenced reads, summed across all input libraries. |
[pre/post]_multi_transcriptome_total_raw_reads_per_filtered_bc | total_reads divided by filtered_bcs_transcriptome_union |
[library_id]_pre_normalization_raw_reads_per_filtered_bc | The mean total reads per spot prior to depth normalization, for the library denoted by library_id |
[library_id]_pre_normalization_cmb_reads_per_filtered_bc | The mean confidently mapped and barcoded (CMB) reads per spot prior to depth normalization, for the library denoted by library_id. |
[library_id]_frac_reads_kept | The fraction of reads that were retained after depth normalization for the library denoted by library_id |
lowest_frac_reads_kept | The lowest fraction of reads retained, corresponding to the library which lost the most reads during normalization. A low value may indicate a large disparity in the initial depth of the input libraries. |
If one or more of the aggregated samples was a Targeted Gene Expression sample, these additional metrics will also appear:
| Metric | Description |
|---|---|
[library_id]_pre_normalization_targeted_reads_per_filtered_bc | The mean targeted reads per spot prior to depth normalization, for the library denoted by library_id |
[library_id]_frac_targeted_reads_kept | The fraction of reads mapped uniquely and confidently to targeted genes that were retained after depth normalization for the library denoted by library_id. This field will be shown instead of the metric [library_id]_frac_reads_kept above |