Calculate Unique Molecular Index Groups

The tool Calculate Unique Molecular Index Groups annotates the mapped reads with a "Unique Molecular Index group ID", that is identical for reads that are determined to belong to the same UMI. The tool can be found in the Toolbox here:

        Tools | QIAseq Panel Expert Tools (Image qiaseq_expert_folder_closed_16_n_p) | QIAseq DNA Panel Expert Tools (Image qiaseqv3_folder_open_16_h_p) | Calculate Unique Molecular Index Groups (Image calculate_bcgroups_16_h_p)

In the first dialog (figure 5.37), select a read mapping of reads that were previously annotated with UMI annotations.

Image readmappingannotatebarcode
Figure 5.37: Select a read mapping made from reads whose UMI was removed and annotated on the sequences.

The grouping of reads into UMI groups works as follows:

  1. The tool groups reads that
    • start at the same position based on the end of the read to which the UMI is ligated (i.e., Read2 for paired data),
    • are from the same strand, and
    • have identical UMIs.
    Groups that contain only one read are called singletons.
  2. The tool then fuzzy merges singleton groups into non-singleton groups, if the UMI of the singleton group can be made into UMI of non-singleton group by introducing a SNP, and if the non-singleton group is the biggest of such group (i.e., if two different introduced SNPs yields two different non-singleton groups, the biggest one is chosen).
  3. Additional merging of singletons and small groups into bigger ones can happen depending on the parameters set for the tool.

It is possible to change the following parameters (figure 5.38):

Image fuzzyparameters
Figure 5.38: Select a read mapping made from reads whose UMI was removed and annotated on the sequences.

Click Next to choose whether to Open or Save the resulting read mapping of reads which now have had a "UMI group ID" annotation.

A report can also be generated. It contains:

Note: When the group sizes (the number of reads in UMI groups) are very large (in most cases more than 10 reads in a UMI group is not desirable), this can indicate problems, such as quality issues with the sample. It can also indicate that the sequencing depth could be reduced.