Quantify miRNA

The Quantify miRNA tool counts and annotates miRNAs using miRBase. It maps the reads against the full precursor miRNA and further processes the mapping to produce expression values for:

The tool performs the mapping using unique search sequences, i.e. collapsed identical reads. This significantly reduces the computational time. The report contains values for both reads and unique search sequences.

The tool will take:

To run the tool, go to:

Toolbox | RNA-Seq and Small RNA Analysis (Image rna_seq_group_closed_16_n_p)| miRNA Analysis (Image mirna_tools_folde_open) | Quantify miRNA (Image mirna_quantification_16_n_p)

First select the trimmed reads as in figure 33.15.

Image quantifymirna1
Figure 33.15: Select the reads.

If the sequencing was performed using Spike-ins controls, the option "Enable spike-ins" can be enabled in the Quality control dialog (figure 33.16), and a spike-ins file can be specified. You can also change the Low expression "Minimum supporting count", i.e., the minimum number of supporting reads for a small RNA to be considered expressed.

Image quantifymirna2
Figure 33.16: Specifying spike-ins is optional, and you can change the threshold under which a small RNA will be considered expressed.

In the annotation dialog, several configurations are available.

In the miRBase annotations section, specify a single reference - miRBase in most cases.

miRBase can be downloaded using the Reference Data Manager under QIAGEN Sets | Reference Data Elements | mirBase (figure 33.17).

Image mirbaserefmanager
Figure 33.17: Download the latest miRBase database in the Workbench.

You can also import miRBase into the CLC Genomics Workbench using Standard Import (Image Next_Folder_16_n_p). The miRBase data file can be downloaded from ftp://mirbase.org/pub/mirbase/CURRENT/miRNA.dat.gz. Select MiRBase (.dat) in the Force import as type menu of the Standard Import dialog. Information about the miRBase dat format is provided in miRBase data file format.

Once miRBase has been selected, click the green plus sign to see the list of species available. It can take a while for all species to load. Species to be used for annotation should be selected using the left and right arrows, and prioritized using the up and down arrows, with the species sequenced always selected as top priority in the list (figure 33.18). The naming of the miRNA will depend on this prioritization.

In addition, it is possible to configure how specific the association between the isomiRs and the reads has to be by allowing mismatches and additional or missing bases upstream and downstream of the isomiR.

Image quantifymirna3
Figure 33.18: Specify and prioritize species to use for annotation, and how stringent the annotation should be.

In the Custom databases, you can optionally add sequence lists with additional smallRNA reference databases, e.g. piRNAs, tRNAs, rRNAs, mRNAs, lncRNAs. An output with quantification against the custom databases can be generated, which can be used for subsequent expression analyses. Reads count towards the reference to which they map best, regardless of which database (miRBase or custom) the reference is from.

Finally, configure the Alignment settings by defining how many "Maximum mismatches" are allowed between the reads and the references, i.e. miRBase and custom databases. Reads matching more than 100 references are ignored.

In the next dialog (figure 33.19), specify the length of the reads used for seed counting. Reads of the specified length, corresponding to the length of mature miRNA (18-25 bp by default, but this parameter can be configured) are used for seed counting. The seed is a 7 nucleotide sequence from positions 2-8 on the mature miRNA. The "Grouped on seed" output table includes a row for every seed that is observed in miRBase together with the expression of the same seed in the sample. In addition, the 20 most highly expressed novel seeds are output in the report.

Image quantifymirna4
Figure 33.19: This dialog defines the length of the reads that will be merged according to their seed.

The tool will output the following expression tables:

The expression tables can be used for subsequent expression analysis tools such as Differential Expression, PCA for RNA-Seq and Create Heat-Map for RNA-Seq. In addition, and depending on the options selected in the last dialog, the tool can output a report and a sequence list of reads that could not be mapped to any references. For a detailed description of the outputs from Quantify miRNA see Quantify miRNA outputs