QIAseq miRNA Library Kits
Quality control summaryThe summary table displays key data points from the Quality control tab. Depending on the selected reference, annotation-specific columns may be omitted.
- Sample name: The name of the sample.
- Reads: The number of reads in the sample data.
- Low numbers could indicate library prep issues.
- UMI Reads: Unique Molecular Indexes are used to join reads with the same amplification origin into UMI reads. This allows for better quantification of the miRNAs by eliminating any library amplification and sequencing bias.
- Avg Q Score (UMI Reads): The average quality score of the UMI reads.
- Numbers less than 30 indicate poor quality library prep or instrument runs.
- Total mapped UMI reads: The total number of UMI reads that were annotated with miRBase or custom piRNA records during the Quantify miRNA analysis step and mapped to the reference sequence during the RNA-Seq Analysis step (see Appendix B).
- miRNA (UMI reads): The number of UMI reads annotated with record from the miRBase database.
- piRNA (UMI reads): The number of UMI reads annotated with records from the piRNA database.
- Protein coding (UMI reads): The number of UMI reads mapped to protein coding genes.
- tRNA total (UMI reads): The number of UMI reads mapped to tRNA genes.
- rRNA total (UMI reads): The number of UMI reads mapped to rRNA genes.
Creation of UMI reads
Detailed QC from the process of creating the UMI reads as single consensus reads, from reads that have the same Unique Molecular Index.
- Input reads: The number of reads in the sample data. This is the same as the 'Reads' column in the Summary.
- Low numbers in any samples could indicate library prep issues.
- Avg Q Score, input reads: The average quality score of the sample data.
- Discarded reads: Reads where the common sequence from the UMI is not found, or where the lengths of the miRNA or UMI do not fulfill the predefined criteria.
- UMI groups: The number of UMI groups. The UMI process identifies similar reads via the index and joins them in a group of reads.
- UMI reads (merged UMI groups): Although indexed as different reads, some UMI reads may originate from the same biological read or fragment and have the same genetic code. In the process of creating UMIs, reads with the same index are grouped. Identical groups are subsequently merged into a Merged UMI group. This final consensus is the 'UMI read' used in downstream analysis. The terms 'UMI read' and 'UMI' are used interchangeably throughout the report.
- Avg Q score (UMI reads): The average quality score of the UMI reads.
- Numbers less than 30 indicate poor quality library prep or instrument runs.
- Avg reads per UMI: The average number of raw reads in each UMI read.
- Should be greater than one.
- UMIs with less than 9 reads: This column together with 'Max reads per UMI' highlights the extreme end of the UMI grouping distribution and should be seen as indications of potential problems in sequencing or library prep. For most applications, the ideal merged UMI group size will be 2-4 reads. Larger UMI groups consume sequencing capacity without providing additional benefits.
- A high percentage is preferable. Numbers less than 90 indicate sample prep issues.
- Max reads per UMI: This indicates the extreme end of the UMI grouping to highlight distribution and potential problems in sequencing.
Quantify miRNA - Annotation records found
- miRBase (species): 'Records in source' indicates the number total number of Precursor miRNAs (pre-miRNAs) in the database used for annotating the features. Note that miRBase records are pre-miRNAs, whereas sample findings indicate the numbers of corresponding mature miRNAs.
For each sample, the number and percentage indicates how many of the database records were seen in the sample. - piRNA (piRNAdb_species): 'Records in source' indicates the total number of records available in piRNAdb for the given species.
For each sample, the number and percentage indicates how many of the database records were seen in the sample. See reference appendix for more information on piRNA version.
Quantify miRNA - Unique search sequences
For annotating the reads with database information, the analysis collapses the reads into unique search sequences. Collapsing identical reads into unique search sequences significantly reduces the number of miRNA reads in the subsequent annotation step and thereby saves computational time. The annotations are subsequently transferred to the UMI reads used in the expression analysis.
- Annotated: Unique search sequences annotated with database records.
- Annotated with miRBase (species): Unique search sequences annotated with miRBase database records.
- Annotated with piRNA (piRNAdb_species): Unique search sequences annotated with piRNA database records.
- Unannotated: Unique search sequences that did not match database records.
- Total: The total number of unique search sequences.
Quantify miRNA - Reads
Distribution of UMI reads annotated with the records from the applied databases. Annotations are transferred from the unique search sequences.
- Annotated: Reads annotated with miRBase or custom piRNA records.
- A low percentage could indicate sample prep issues or contamination.
- Annotated with miRBase (species): Reads annotated with miRBase database records. Note that miRBase annotations include reads annotated outside mature regions, which are ignored in the counts and differential expression analyses. This can result in a higher number shown here compared to the number of features in your differential expression analysis or in expression value downloads.
- Annotated with piRNA (piRNAdb_species): Reads annotated with piRNA database records.
- Unannotated: Reads that did not match database records.
- Total: The number of reads used in the Quantify miRNA analysis step.
This value may differ from the number of output UMI reads generated by the Create UMI Reads for miRNA analysis step (UMI reads (merged UMI groups) in the report section Creation of UMI reads). The Create UMI Reads for miRNA tool introduces N's at positions where merged reads disagree. As a first filtering step, the Quantify miRNA tool discards any UMI reads containing N's, and these are therefore not included in the Total count.
Spike-in quality control
This section is available if the Spike-ins option was selected in the Align and Count dialog.
- Spike-ins detected: The number of spike-ins detected relative to the spike-ins used.
- UMI reads mapped to spike-ins: The number of UMI reads that mapped to the detected spike-ins.
- % of total UMI reads: Percentage of UMI reads that mapped to spike-ins.
RNA-Seq Analysis - Reads
- Input reads: Number of UMI reads used as input for the RNA-Seq analysis step.
- Mapped reads: Total number of mapped UMI reads.
- Mapped to gene: Number of UMI reads that mapped partly or entirely within a gene.
- Mapped to exon: Number of UMI reads that mapped entirely within an exon or to an exon-exon junction.
- Mapped to intergenic region: Number of UMI reads that mapped partly or entirely to intergenic regions.
RNA-Seq Analysis - Biotype distribution
The table summarizes the biotype distribution of reads used as input for the RNA-Seq analysis step, i.e., reads not annotated with miRBase or piRNA records during the Quantify miRNA step. Biotype names and classifications follow Ensembl definitions: