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10x Genomics
Chromium Single Cell Gene Expression

Cell Multiplexing Metrics

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.

Headline metrics

These metrics describe the overall quality of the multiplexing experiment.

MetricDescription
Estimated number of cell-associated barcodesNumber of cell-associated barcodes called as containing one or more cells.
Number of samples assigned at least one cellNumber 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 sampleNumber of cells assigned to a sample. Only cell-associated barcodes assigned exactly one CMO were assigned to a sample.
Singlet capture ratioRatio 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 multipletsFraction of cell-associated barcodes that were assigned more than one CMO and hence identified as multiplets.
Median CMO UMIs per cellMedian number of CMO UMIs captured per cell-associated barcode assigned exactly one CMO.

Sample Assignment Metrics

These metrics are reported per physical library and describe the quality of the assignment of barcodes to multiplexed samples.

MetricDescription
Estimated number of cell-associated barcodesNumber of cell-associated barcodes called as containing one or more cells.
Mean reads per cell-associated barcodeThe total number of sequenced read pairs divided by the number of cell-associated barcodes.
Samples assigned at least one cellNumber 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 sampleNumber of cells assigned to a sample. Only cell-associated barcodes assigned exactly one CMO were assigned to a sample.
Cell-associated barcodes identified as multipletsCell-associated barcodes that were assigned more than one CMO and hence determined to be multiplets.
Cell-associated barcodes not assigned any CMOsCell-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 barcodeMedian number of CMO UMIs captured per cell-associated barcode.

Sequencing Metrics

These metrics are reported per Fastq ID and describe the quality of the sequencing runs for Multiplexing libraries.

MetricDescription
Number of readsTotal number of read pairs sequenced during this run.
Number of short reads skippedTotal 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 barcodesFraction of cell barcode bases with Q-score >= 30, excluding very low quality/no-call (Q <= 2) bases from the denominator.
Q30 UMIFraction of UMI bases with Q-score >= 30, excluding very low quality/no-call (Q <= 2) bases from the denominator.
Q30 RNA readFraction of RNA Read bases with Q-score >= 30, excluding very low quality/no-call (Q <= 2) bases from the denominator.

Metrics Per Physical Library

These metrics are reported per Physical Library and describe multiplexing library quality.

MetricDescription
Number of readsTotal number of read pairs that were assigned to this library.
Valid barcodesFraction of reads with barcodes that are present in the whitelist after barcode correction.
Valid UMIsFraction of reads with valid UMIs; i.e. UMI sequences that do not contain Ns and that are not homopolymers.
Fraction reads in cell-associated barcodesThe fraction of valid-barcode, valid-UMI, recognized multiplexing-barcode reads with cell-associated barcodes.
Mean reads per cell-associated barcodeThe total number of sequenced read pairs divided by the number of cell-associated barcodes.
Fraction CMO readsFraction of reads that contain a recognized CMO sequence.
Fraction CMO reads usableFraction of read pairs that contain a recognized CMO sequence, a valid UMI, and a cell-associated barcode
Fraction unrecognized CMOFraction of read pairs with an unrecognized CMO sequence.
Fraction reads from multipletsAmongst all sequenced read pairs, fraction with a cell-barcode identified as a multiplet.

Metrics Per CMO

These metrics describe the quality of results for individual CMOs.

MetricDescription
Fraction reads in cell-associated barcodesAmongst all reads with a valid barcode, valid UMI, and this particular CMO sequence, fraction arising from cell-containing partitions.
Cells assigned to CMONumber of cells assigned this particular CMO (and only this CMO).
CMO signal-to-noise ratioComputed as the difference between labeled and unlabelled mean CMO counts (log scale) divided by the standard deviation.

Cell Assignment Metrics per Sample

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.

MetricDescription
Cells assigned to this sampleNumber 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 samplesNumber 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 CMOsCell-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 multipletsCell-associated barcodes that were assigned more than one CMO and hence determined to be multiplets.

Cell Statistics per Library

These metrics about cmo and sample assignment for cells will be reported per physical library.

MetricDescription
Cells assigned to a sampleNumber 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 CMOsCell-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 multipletsCell-associated barodes that were assigned more than one CMO and hence determined to be multiplets.