Validate QIAseq Read Structure (beta)

The Validate QIAseq Read Structure (beta) tool has the ability to analyze and validate the read structure for selected QIAseq panels and kits by predicting changes between UMI, common/adapter sequence and biological sequence based on nucleotide contributions for the four DNA nucleotides in the read. The tool takes a Graphical Report from QC for Sequencing Reads as input.

Validate QIAseq Read Structure (beta) predicts segments in the read by analyzing the changes in Standard Deviation (SD) of the nucleotide contributions in a sliding window throughout the read. If the nucleotide contribution for one of the four nucleotides in a position next to the window is significantly different from the nucleotide distribution inside the window, a potential change in read structure is found. The read structure prediction sensitivity parameter determines how many standard deviations the position next to the window should be from the mean of the window, in order to be significantly different.

When read segments have been predicted, the tool compares the predicted segments to the expected read structure. If all segments of the expected read structure are found in the sample, the read will pass the comparison. For paired end reads, R1 and R2 reads are analyzed and compared individually and the sample will pass if both of the reads pass.

The validation of read segments is based on three different criteria:

The tool can be found in the Toolbox here:

        Toolbox | Biomedical Genomics Analysis (Image biomedical_folder_closed_16_n_p) | QIAseq Tools (Image qiaseq_expert_folder_closed_16_n_p) | Validate QIAseq Read Structure (beta) (Image qc_for_readstructure_16_h_p)

Double-click to run the tool.

Select one or more Graphical Report produced by QC for Sequencing Reads from the navigation area and add them to the list on the right hand side of the dialog using the arrows. Remember to enable Batch when analyzing multiple samples.

In the next wizard step select the QIAseq protocol used for generating the reads in the 'Expected QIAseq read structure' drop-down menu or select 'Unknown' if the kit is not supported. The tool will be preconfigured with optimal parameters for the different kits, however, sequencing is not an exact science and you might have to adjust the parameters to obtain the expected result. It is possible to adjust a number of parameters, see figure 5.7:

Image validate_read_structure_wizard
Figure 5.7: Parameter wizard step of the Validate QIAseq Read Structure (beta)