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 at:

        Toolbox | Biomedical Genomics Analysis (Image biomedical_folder_closed_16_n_p) | UMI Tools (Image qiaseqv3_folder_open_16_h_p) | Calculate Unique Molecular Index Groups (Image calculate_bcgroups_16_h_p)

In the first dialog (figure 4.2), select a read mapping of reads that were previously annotated with UMI annotations.

Image readmappingannotatebarcode
Figure 4.2: 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 depending on which read structure was used in the Remove and Annotate with Unique Molecular Index tool, (If the UMI was removed from the start of read 2 using the Remove and Annotate with Unique Molecular Index tool, this tool considers grouping reads where the start of read 2 map to the same position)
    • are from the same strand, and
    • have identical UMIs.
    The tool then merges smaller groups into larger groups if
  2. Their start positions are sufficiently close as defined by the Window size parameter.
  3. Their UMIs are similar enough as defined by the Fuzzy match Unique Molecular Indices parameter.

    Merging is only done if the larger group is sufficiently large compared to the smaller group as defined by the parameters described below. If a smaller group can be merged into multiple larger groups that are equally good in terms of similarity of UMI and start position as well as group size, the group will not be merged.

It is possible to change the following parameters (figure 4.3):

Image fuzzyparameters
Figure 4.3: 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 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.

This report can be used together with the Combine Reports tool (see http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Combine_Reports.html)