Sequencing:

Number of Reads

• Total number of read pairs that were assigned to this library in demultiplexing.

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 cell-barcode, valid UMI reads that had a non-unique (cell-barcode, UMI, gene). This metric was called 'cDNA PCR Duplication' in versions of Cell Ranger prior to 1.2.

Q30 Bases in Barcode

• Fraction of cell 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 1 for the Single Cell 3' v1 chemistry and Single Cell 5' paired end, Read 2 for the Single Cell 3' v2/v3 chemistry and Single Cell 5' R2-only).

Q30 Bases in UMI

• Fraction of UMI bases with Q-score >= 30, excluding very low quality/no-call (Q <= 2) bases from the denominator.

Median UMI Counts per Cell

• The median number of UMI counts per %s cell-associated barcode.

Mapping:

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 when include-introns=false. 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.

The plot shows the count of filtered UMIs mapped to each barcode. Barcodes are not determined to be cell-associated strictly based on their UMI count. Instead, they could be determined based on their expression profile, or removed via Protein Aggregate Detection and Filtering and/or High Occupancy GEM Filtering. Therefore, some regions of the graph contain both cell-associated and background-associated barcodes. The color of the graph in these regions is based on the local density of barcodes that are cell-associated.

This plot shows the Median Genes per Cell as a function of downsampled sequencing depth in mean reads per cell, up to the observed sequencing depth. The slope of the curve near the endpoint can be interpreted as an upper bound to the benefit to be gained from increasing the sequencing depth beyond this point.

This plot shows the Sequencing Saturation metric as a function of downsampled sequencing depth (measured in mean reads per cell), up to the observed sequencing depth. Sequencing Saturation is a measure of the observed library complexity, and approaches 1.0 (100%) when all converted mRNA transcripts have been sequenced. The slope of the curve near the endpoint can be interpreted as an upper bound to the benefit to be gained from increasing the sequencing depth beyond this point. The dotted line is drawn at a value reasonably approximating the saturation point.