QIAseq Stranded RNA Library Kits (FastSelect RNA Library Kit, Stranded mRNA/Stranded Total RNA Library Kits), QIAseq UPXome Kit, and all third-party kits
Quality control summaryThe summary table presents key data points from the Quality Control Report.
- Sample name: The name of the sample.
- Reads: The number of reads in the sample data.
- Low numbers in any samples indicate failed library prep.
- Trimmed reads: Quality trimming is performed on the input reads.
- Mapped/Mapped in pairs: The number of reads that mapped to the reference. If the input was paired reads, this value will be the number of reads that mapped in pairs, i.e reads that mapped as broken pairs are left out.
- A low percentage can be an indication that the wrong reference was selected, that data is of poor quality, or a sign of sample contamination.
- Mapped to total rRNA: Percentage of reads mapped to ribosomal RNA (rRNA) or mitochondrial ribosomal RNA (MtrRNA).
- If rRNA was not depleted, percentages can vary from 10%-30%.
- If rRNA was depleted, percentages should be low, about or less than 1%.
- Samples not depleted of rRNA or samples with higher percentages can still be used for differential expression, but expression values such as TPM and RPKM may not be comparable to those of other samples. To troubleshoot the issues in future experiments, check for rRNA depletion prior to library preparation. Also, if an rRNA depletion kit was used, check that the kit matches the species being studied.
Trimming
Metrics for trimming of raw input reads. If the number of reads and average read length decrease substantially after trimming, it indicates poor read quality.
- Reads before trim: The number of raw reads prior to trimming. This value is also shown in the 'Reads' column in the summary table.
- Avg length before trim: The average read length before trimming.
- Reads after trim: The number of raw reads after trimming. This value is also shown in the 'Trimmed reads' column in the summary table.
- Avg length after trim: The average read length after trimming.
Spike-in quality control
This section is available if the Spike-ins option was selected in the Align and Count dialog.
- Number of spike-ins detected: The number of spike-ins detected relative to the spike-ins used.
- R2: Correlation of expected and sequenced spike-ins using the Pearson Correlation coefficient
- When samples have a poor correlation (R2 < 0.8) between known and measured spike-in concentrations, it indicates problems with the spike-in protocol or a more serious problem with the sample.
- Reads mapped to spike-ins: The number of reads that mapped to the detected spike-ins.
- If fewer than 10,000 reads mapped to spike-ins, consider using more spike-in mix in future experiments.
- Lower limit of detection (attomoles/ul): Spike-ins concentration measurement. The lower limit of detection is the lowest concentration spike-in to which at least 3 reads map. This provides a rough estimate of the minimal concentration of mRNA that can be detected in this sample.
Mapping statistics
Metrics for the step of mapping reads to the reference genome.
- Reads: The number of reads used as input for the mapping step.
- Paired (yes/no): Indication of whether the input reads are paired reads. This should fit with the applied protocol.
- Reads mapped (in pairs): The percentage of the reads that were mapped to the selected reference. Excludes both reads that were ignored due to wrong strand and reads that could not be mapped to the reference.
- Strand-specific setting: Read direction of reads. This should fit the applied protocol.
- Forward % of reads mapped: Percent of reads mapped in the forward direction.
- Reverse % of reads mapped: Percent of reads mapped in the reverse direction.
- Ignored reads % (wrong strand): The percentage of reads that were ignored because the did not map to the strand defined by the strand-specific setting.
- If percentages are greater than 20-25% that the wrong strand protocol may have been used in library prep.
Mapped by type
This section describes the relative mapping of the UMI reads in terms of the type of target.
- Mapped to gene: Percentage of reads that map to genes.
- Mapped to gene, intron: Percentage of reads that mapped partly or entirely within an intron.
- Mapped to gene, exon: Percentage of reads that mapped entirely within an exon or to an exon-exon junction.
- Mapped to intergenic region: Percentage of reads that mapped partly or entirely between genes.
Biotype Distribution
Metrics covering the biotype distribution. The plot and the table show which biotypes are found in the samples and at which relative abundance. The names and classification of the biotypes is based on the Ensembl definitions found here:
http://www.ensembl.org/info/genome/genebuild/biotypes.html.
Taxonomic profile of unmapped reads
Taxonomic profiling is performed for samples with a high level of unmapped reads as this can indicate sample contamination. If all samples have low levels of unmapped reads, this section will be empty. Plot and table show the relative abundance at phylum level. Reads that map equally well to two or more phyla are assigned to the common ancestor (kingdom level).
Taxonomic profiling summary
Information about which taxonomic levels were found in the data sample and how many different taxa were found on each level.
- Kingdom
- Phylum
- Total reads: The number of reads used as input for this step, i.e. reads that did not map the reference genome.
- Classified reads: The number of reads that mapped to the taxonomic profiling database.
- Unclassified reads: The number of reads that mapped to neither the reference nor the taxonomic profiling database. These are reads of unknown origin. If this number constitutes a significant portion of the input reads, it may be due to the selection of the wrong reference.