Trim Primers of Mapped Reads

The tool Trim Primers of Mapped Reads removes the primer parts of mapped reads, as they reflect the primer that was added and not the actual sample. Note that the tool will also remove any insertion located immediately after the primer. The tool also removes artifacts due to mispriming events, happening when a primer binds an off-target location and thus amplifies an off-target sequence.

The tool can be found in the Toolbox here:

        Toolbox | Biomedical Genomics Analysis (Image biomedical_folder_closed_16_n_p) | Biomedical Utility Tools (Image utilities_closed_16_n_p) | Trim Primers of Mapped Reads (Image trim_primers_v3_16_h_p)

In the first dialog (figure 6.10), select a read mapping.

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Figure 6.10: Select a read mapping.

In the second dialog (figure 6.11), select the primer annotation track that was provided with the QIAseq Panel. This track contains the original primers and their intended primer locations.

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Figure 6.11: Select the primer annotation track specific to the panel, and add the parameters needed to deal with the type of reads you are working with.

In addition, set the following parameters:

If one read in a UMI group runs past the primer it overlaps, it means that all reads in that group were not created from that primer. If this happens, then the tool will not unalign any reads in this UMI group.

In the Remove mispriming artifacts dialog (figure 6.12), you can specify a Primer mispriming events track containing the predicted off-target priming locations of the original primers. Mispriming tracks can be generated by the tool Identify Mispriming Events (see section 6.3) and are available for each QIAseq panel from the Reference Data Manager.

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Figure 6.12: Select a mispriming track specific to the panel, and configure the associated parameters if needed.

In addition, set the following parameters:

In the Post-filtering dialog (figure 6.13), when the "Remove short reads" option is enabled, reads with an alignment length shorter than the value specified after primer trimming will be removed from the mapping.

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Figure 6.13: Post-filtering parameters.

The tool will output a trimmed read mapping. In addition, as seen in figure 6.14, you can choose to output off-target primers and/or regions tracks with counts.

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Figure 6.14: Outputs options.

If a mispriming track was specified in the earlier step, it is possible to output two additional tracks containing respectively region and primer mispriming statistics: