# Single-Cell RNA ### Barcode Rank Plot The barcode rank plot (often referred to as the “knee plot”), orders barcodes based on the number of molecules associated with them. Typically, the cell barcodes are concentrated at the top of the rank plot, whereas the background barcodes are concentrated in the lower portion of the plot. The purpose of the cell calling is to find a point in the first “knee” area that separates the cells from the background. A more negative slope and a poorly defined knee can be indicative of lower quality samples. It is not recommended to call cells too far down into the transition zone (area between first knee and second knee) as this risks adding compromised cells and background noise. * Homogenous sample types and cells should be called towards the upper part of the transition zone * More heterogenous sample types should be called ⅓ or ½ way down the transition zone
### Single Cell RNA Metrics | Metric | Description | | ------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Input Reads | Total number of reads that were successfully sequenced for each library and demultiplexed during BCL-convert. This includes reads missing barcodes, reads with exactly matching barcodes, reads with corrected barcdoes, and reads with non-matching barcodes. | | % Barcoded Reads | Percentage of total input reads with a cell barcode that match the whitelist. This includes reads with exactly matching barcodes and reads with corrected barcodes (either an exact match or within one edit distance away for each of the four barcode tiers). Typically 85-95% for cell lines and RNA-rich cells or 70-90% for nuclei and cells with low RNA content. | | % Mapped to Transcriptome |

Percentage of total barcoded reads that are mapped to the transcriptome and are included in downstream analysis (barcode matching, counting, etc.). This includes:

| | % Sequencing Saturation | Represents the probability that adding additional sequenced read will have already been observed. The higher the sequencing saturation, the less benefit of uncovering new information with additional sequencing reads. | | Passing Cells | Total unique barcodes that pass the count threshold as set by the ratio, inflection, or forced cell calling approaches. | | % Reads in Passing Cells | Number of reads in passing barcodes divided by the total number of barcoded reads in all cells | | Mean Reads per Cell | Total input gene expression reads divided by the number of passing cells. Generally, should fall within range of 20,000-40,000 reads per (called) cell at minimum to ensure sufficient read depth. If mean reads per cell is lower than 20,000, it might be necessary to sequence at a higher read depth. | | Median Molecules per Cell | The median number of captured RNAs per passing barcode. A molecule represents an individual transcript that was captured onto a PIP, was converted into a cDNA library molecule (with one or more daughter molecules during fragmentation), sequenced, then properly barcoded, mapped, and IMI-corrected. | | Median Genes per Cell | Median number of unique gene assignments observed across all molecules in the passing cell fraction | | Genes Detected | Total unique genes detected in passing cells | ### Mapping Metrics | Metric | Description | | ------------------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | % Mapped to Transcriptome |

Percentage of total barcoded reads that are mapped to the transcriptome and are included in downstream analysis (barcode matching, counting, etc.). This includes:

| | % Mapped to Genome |

Percentage of total barcoded reads that are:

Filtered read categories are discarded from downstream analysis (barcode matching, counting, etc.)

| | % Exon Matching | Percentage of total barcoded reads that map to exons in genes | | % Intron Matching | Percentage of total barcoded reads that map to introns in genes | | % Mitochondrial | Percentage of total barcoded reads that map to mitochondria | | % Antisense | Percentage of total barcoded reads that map to the opposite strand of a gene | | % Ambiguous | Percentage of total barcoded reads that map to multiple genes equally well | | % Low MAPQ | Percentage of total barcoded reads that below the MAPQ threshold (default: 4). These reads map to multiple positions in the genome. This metric is not included if feature counting is enabled. | | % Non-Matching | Percentage of total barcoded reads that do not map to any genes | ### Feature Metrics This section is only available for analyses with feature counting enabled. | Metric | Description | | --------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | Input Feature Reads | Total number of input feature reads | | % Barcoded Feature Reads | Percentage of input feature reads with barcode matching whitelist after error-correction. This includes reads with exactly matching barcodes and reads with corrected barcodes (either an exact match or within 1 edit distance away for each of the four barcode tiers) | | % Feature Matching Reads | Percentage of total barcoded reads that match to a feature reference sequence | | % Feature Reads in Cells | Percentage of total barcoded feature reads that are reads in passing cells | | % Feature Matching Reads in Cells | Percentage of feature matching CRISPR reads that are found in passing cells | | % Cells with Features | Percentage of passing cells with at least 1 CRISPR read count detected | | Mean Feature Reads per Cell | Mean number of feature reads per passing cell | | Median Feature Molecules per Cell | Median number of feature reads observed per passing cell. Note: CRISPR and feature read applications use read-based counting in DRAGEN because they do not contain IMIs | | Median Features per Cell | Median number of unique guide RNA types (or feature types) present per passing cell | | Features Detected | Total number of features observed across all cells in the dataset | ### Guide Assignment Metrics | Metric | Description | | --------------------------- | ------------------------------------------------------------------- | | Passing Cells with Features | Total number of passing cells containing at least one feature count | | Cells with ≥ 1 Guide | Number of feature-positive cells positive for at least 1 guide | | Cells with ≥ 2 Guides | Number of feature-positive cells positive for 2 or more guides |