Space Ranger2.0, printed on 12/18/2024
Space Ranger pipelines output key metrics in text format. Below are the definitions of the reported metrics.
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 1 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 >= 30, excluding very low quality/no-call (Q <= 2) bases from the denominator. |
Q30 Bases in RNA Read | Fraction of RNA read bases with Q-score >= 30, excluding very low quality/no-call (Q <= 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 >= 30, excluding very low quality/no-call (Q <= 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 | 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 >= 10 UMIs | Number of genes with at least 10 filtered UMIs from tissue-associated barcodes. These genes are used to calculate per-gene enrichments. |
A Targeted Spatial Gene Expression library has additional metrics specific to targeting.
| Metric | Description |
|---|---|
Analyzed Reads | Fraction of the reads used for mapping and analysis. This fraction will be less than 100% when the sequencing depth exceeds 15,000 Mean Reads Under Tissue per Spot. See Targeted Subsampling. |
Mean Targeted Reads per Spot | The number of targeted reads, both under and outside of tissue, divided by the number of barcodes associated with a spot under tissue. |
Number of Targeted Genes | Number of targeted genes as specified in the input target panel file. |
Median Targeted Genes per Spot | The median number of targeted genes detected per tissue-associated barcode. Detection is defined as the presence of at least 1 UMI count. This metric will appear instead of the metric Median Genes per Spot above. |
Median Targeted UMI Counts per Spot | The median number of targeted UMI counts per tissue-associated barcode. This metric will appear instead of the metric Median UMI Counts per Spot above. |
Targeted Sequencing Saturation | The fraction of targeted reads originating from an already-observed targeted UMI. This is a function of library complexity and sequencing depth. More specifically, this is the fraction of confidently mapped, valid spot-barcode, valid targeted UMI reads that had a non-unique (spot-barcode, UMI, gene). This metric will appear instead of the metric Sequencing Saturation above. |
Total Targeted Genes Detected | The number of targeted genes with at least one UMI count in any spot. This metric will appear instead of the metric Total Genes Detected above. |
Number of Targeted Genes | Number of targeted genes as specified in the input target panel file. |
Number of Non-Targeted Genes | Number of genes in the reference genome that are not targeted. |
Number of Targeted Genes >= 10 UMIs | Number of targeted genes with at least 10 UMIs in spot-associated barcodes. These genes are then considered in the calculation of per-gene enrichments. |
Number of Non-Targeted Genes >= 10 UMIs | Number of non-targeted genes with at least 10 UMIs in spot-associated barcodes. These genes are then considered in the calculation of per-gene enrichments. |
Number of Enriched Targeted Genes | Number of targeted genes that are classified as enriched as a result of having high mean reads per UMI. Only genes with at least 10 spot-associated UMIs can be enriched. See Targeted Gene Expression Algorithms. |
Number of Enriched Non-Targeted Genes | Number of non-targeted genes that are classified as enriched as a result of having high mean reads per UMI. Only genes with at least 10 spot-associated UMIs can be enriched. See Targeted Gene Expression Algorithms.. |
Mean Reads per UMI per Targeted Gene | Mean number of reads per UMI per targeted gene, averaged across all targeted genes with at least 10 spot-associated UMIs. |
Mean Reads per UMI per Non-Targeted Gene | Mean number of reads per UMI per non-targeted gene, averaged across all targeted genes with at least 10 spot-associated UMIs. |
Reads Mapped Confidently to the | Fraction of Reads Mapped Confidently to the Targeted Transcriptome. |
Reads Mapped Confidently to the | 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. This fraction is computed separately for targeted and non-targeted gene categories. |
Fraction of Targeted Genes Enriched | Fraction of Targeted Genes with >= 10 spot-associated UMIs that are enriched. |
High Fraction of Non-Targeted Genes Enriched | High Fraction of Non-Targeted Genes Enriched. |