The cellranger multi command computes sequencing quality and application metrics on each supported library, which currently are Gene Expression, Multiplexing Capture, VDJ, Antibody Capture, CRISPR Guide Capture, and Custom (user-defined assays). These metrics will be computed and displayed only when one of these library types was used in the assay. This page describes Multiplexing library metrics for QC of the library prep and sequencing of Multiplexing Capture libraries, which appear in the metrics summary CSV and on the web summary ("Multiplexing" tab).
These metrics describe the overall quality of the multiplexing experiment.
| Metrics | Description |
|---|---|
Estimated number of cell-associated barcodes | Number of cell-associated barcodes called as containing one or more cells. |
Number of samples assigned at least one cell | Number of samples to which at least one cell was assigned. Only cell-associated barcodes assigned exactly one CMO were assigned to a sample. |
Cells assigned to a sample | Number of cells assigned to a sample. Only cell-associated barcodes assigned exactly one CMO were assigned to a sample. |
Singlet capture ratio | Ratio between the number of singlets (i.e. cell-associated barcodes assigned exactly one CMO) obtained and the number of singlets expected in this experiment according to Poisson statistics. |
Cell-associated barcodes identified as multiplets | Fraction of cell-associated barcodes that were assigned more than one CMO and hence identified as multiplets. |
Median CMO UMIs per cell | Median number of CMO UMIs captured per cell-associated barcode assigned exactly one CMO. |
These metrics are reported per physical library and describe the quality of the assignment of barcodes to multiplexed samples.
| Metric | Description |
|---|---|
Estimated number of cell-associated barcodes | Number of cell-associated barcodes called as containing one or more cells. |
Mean reads per cell-associated barcode | The total number of sequenced read pairs divided by the number of cell-associated barcodes. |
Samples assigned at least one cell | Number of samples to which at least one cell was assigned. Only cell-associated barcodes assigned exactly one CMO were assigned to a sample. |
Cells assigned to a sample | Number of cells assigned to a sample. Only cell-associated barcodes assigned exactly one CMO were assigned to a sample. |
Cell-associated barcodes identified as multiplets | Cell-associated barcodes that were assigned more than one CMO and hence determined to be multiplets. |
Cell-associated barcodes not assigned any CMOs | Cell-associated barcodes that either (i) did not have enough CMO molecules above background or (ii) could not be confidently assigned to a singlet or multiplet state. |
Median CMO UMIs per cell-associated barcode | Median number of CMO UMIs captured per cell-associated barcode. |
These metrics are reported per Fastq ID and describe the quality of the sequencing runs for Multiplexing libraries.
| Metrics | Description |
|---|---|
Number of reads | Total number of read pairs sequenced during this run. |
Number of short reads skipped | Total number of read pairs that were skipped by the pipeline because they do not satisfy the minimum length requirements (for example Read-1 less than 26 bases in 3' v2 or 3' v3 or 5'). |
Q30 barcodes | Fraction of cell barcode bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q less than or equal to 2) bases from the denominator. |
Q30 UMI | Fraction of UMI bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q less than or equal to 2) bases from the denominator. |
Q30 RNA read | Fraction of RNA Read bases with Q-score greater than or equal to 30, excluding very low quality/no-call (Q less than or equal to 2) bases from the denominator. |
These metrics are reported per Physical Library and describe multiplexing library quality.
| Metric | Description |
|---|---|
Number of reads | Total number of read pairs that were assigned to this library. |
Valid barcodes | Fraction of reads with barcodes that are present in 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. |
Fraction reads in cell-associated barcodes | The fraction of valid-barcode, valid-UMI, recognized multiplexing-barcode reads with cell-associated barcodes. |
Mean reads per cell-associated barcode | The total number of sequenced read pairs divided by the number of cell-associated barcodes. |
Fraction CMO reads | Fraction of reads that contain a recognized CMO sequence. |
Fraction CMO reads usable | Fraction of read pairs that contain a recognized CMO sequence, a valid UMI, and a cell-associated barcode. |
Fraction unrecognized CMO | Fraction of read pairs with an unrecognized CMO sequence. |
Fraction reads from multiplets | Amongst all sequenced read pairs, fraction with a cell-barcode identified as a multiplet. |
These metrics describe the quality of results for individual CMOs.
| Metric | Description |
|---|---|
Fraction reads in cell-associated barcodes | Amongst all reads with a valid barcode, valid UMI, and this particular CMO sequence, fraction arising from cell-containing partitions. |
Cells assigned to CMO | Number of cells assigned this particular CMO (and only this CMO). |
CMO signal-to-noise ratio | Computed as the difference between labeled and unlabelled mean CMO counts (log scale) divided by the standard deviation. |
These metrics about sample assignment for cells will be reported per physical library for each multiplexed sample. In general, the metrics will be the same between libraries from the same gem well since cells and sample assignments are only called once per gem well. Although these metrics are reported per library, usually gene-expression information is used to call cells; and multiplexing capture is used to differentiate samples.
| Metric | Description |
|---|---|
Cells assigned to this sample | Number of cells assigned to this sample. In the multiplexing case, only cell-associated barcodes assigned exactly one CMO can be assigned to a sample. |
Cells assigned to other samples | Number of cells assigned to other samples. Only cell-associated barcodes assigned exactly one CMO can be assigned to a sample. |
Cell-associated barcodes not assigned any CMOs | Cell-associated barcodes that either (i) did not have enough CMO molecules above background or (ii) could not be confidently assigned to a singlet or multiplet state. |
Cell-associated barcodes identified as multiplets | Cell-associated barcodes that were assigned more than one CMO and hence determined to be multiplets. |
These metrics about CMO and sample assignment for cells will be reported per physical library.
| Metric | Description |
|---|---|
Cells assigned to a sample | Number of cells assigned to a sample. Note: For a multiplexed analysis, only cell-associated barcodes assigned exactly one CMO are assigned to samples. |
Cell-associated barcodes not assigned any CMOs | Number of cells assigned to a sample. Note: For a multiplexed analysis, only cell-associated barcodes assigned exactly one CMO are assigned to samples. |
Cell-associated barcodes not assigned any CMOs | Cell-associated barcodes that either (i) did not have enough CMO molecules above background or (ii) could not be confidently assigned to a singlet or multiplet state. |
Cell-associated barcodes identified as multiplets | Cell-associated barcodes that were assigned more than one CMO and hence determined to be multiplets. |