The file called library_{libIndex2}.report.html contains the summary report at the library level, i.e. sequencing, barcode and demultiplexing information for all samples processed in one run of the Quantum Scale RNA kit.
This table gives the barcode matching statistics for the full library. Reads that fail barcode matching are not assigned to any sample.
Barcode Pass: Reads for which all expected barcodes were found. Includes TooShortError which are reads with less than 16 (min_length) bases of RNA sequence after adapter and Poly-A trimming. These are assigned to their sample based on RT barcode and included in "Total Sample Reads", but are filtered before genome alignment
Barcode Error: Reads which were filtered because at least one barcode could not be found or matched against the expected sequences (whitelist). These reads are excluded from all further analysis
Reads per Sample shows the number of reads assigned to each sample based on the RT (sample) barcode. These read counts are before adapter trimming, alignment and duplicate filtering.
Plate-maps showing the total unique transcript counts for each RT barcode
The files called {SampleName}.{LibraryName}.report.html contain the summary report for a single sample, i.e. the set of RT wells (Sample Barcodes) from a Scale RNA dataset assigned to one sample in samples.csv. It shows read, cell and barcode level summary metrics and plots for sample QC. Note that sample here could refer to a whole ScalePlex pool.
Total Sample Reads: The number of reads assigned to the sample based on the RT (sample) barcode, after barcode error reads are removed. This is before adapter trimming, alignment and duplicate filters
Passing Sample Reads: Reads passing pre-alignment filters, specifically RNA sequence length after Poly-A trimming ("Too Short Error")
Reads Mapped to Genome: The fraction of Passing Reads that are aligned anywhere to the genome. This includes multimapping reads
Passing Read Alignments: The fraction of mapped reads retained after alignment filtering; specifically excluding multimappers to more than 6 (starMaxLoci) loci
Reads Mapped to Transcriptome: The fraction of Passing Read Alignments that match one or more annotated genes (exon or intron, in sense direction)
Exonic Reads: The fraction of Reads Mapped to Transcriptome overlapping an exon in the sense direction
Antisense Reads: The fraction of Passing Read Alignments overlapping an exon in the antisense direction (opposite from gene annotation)
Mitochondrial Reads: The fraction of reads mapping to the mitochondrial genome (chrM)
Saturation: The overall sequencing saturation level of this sample; defined as 1 - (UniqueReads / TotalReads) on Reads Mapped to Transcriptome
Average Trimmed Read Length: The average length of RNA reads after adapter and Poly-A trimming
Average Mapped Length: The average length of read alignments to the genome (after softclipping)
Mapped Mismatch Rate: The mismatch rate between mapped reads and the genome (variants and sequencing errors)
Note: All numbers in this table depend on the number of cells called for the sample. Before interpreting these numbers, check the Barcode Rank plot to confirm that it matches expectations.
Cells called: The number of cell barcodes passing filters to be called as a cell
Reads per cell: The overall number of Total Sample Reads divided by the number of cells called
Median Unique Transcript Counts per cell: The median number of transcripts detected per cell; i.e. unique reads mapped to the transcriptome
Median Genes per cell: The median number of unique genes detected per cell
Reads in Cells: The fractions of transcriptome reads that belong to a cell rather than a background barcode
This shows the unique transcript counts for each cell-barcode, sorted from high to low. Each point along the line indicates the proportion of barcodes called as cells (as opposed to background) with that transcript count.
This shows a statistical estimate for the unique transcript counts per cell that would be observed at different shallower sequencing levels for the sample ("Total Sample Reads")
This scatterplot shows the number of unique genes detected for each cell-barcode relative to the reads for that barcode, separating cells from background barcodes.
This scatterplot shows the number of reads vs. the sequencing saturation (1 - (UniqueReads / TotalReads)) for each cell-barcode.
The plot on the left show the number of cells called with each sample (RT) barcode.
The plot of the right show the median unique transcript counts for cells with each barcode.
A breakdown of other barcodes is in the library report.