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Overview

The QC Report is a self-contained HTML page that you should be able to open with any common modern browser:

info

Chrome >= 80
Firefox >= 113
Edge >= 80
Safari >= 16.4
Opera >= 67

The QC Report collects and makes easily accessible a summary of several run metrics, grouped in sections. Each section contains a different dashboard to display summary plots. The interactive dashboards display additional details for each data point when hovering the mouse over it.

The QC Report is divided in two parts:

1. A header with the sample information, main metrics and a link to contact our support.

QC Report header

  • Number of Cells: cells that have been called by pixelator.

  • Avg. Reads Usable per Cell: average number of reads that correspond to molecules in each edge of the cell graph.

  • Median Antibody Molecules per Cell: median number of antibody molecules (or edges) per cell discovered.

2. A body and a footer with the different sections and links to our social media.

QC Report body

Sections

Although we show the content of the body collapsed in the figure above, the different sections contain specific information you may be interested in.

  • Parameters: specific parameters for the run (or all parameters if selected)
  • Cells: cell calling and general metrics at the cell level
  • Cell annotations: cell annotations into the major PBMC lineages
  • Antibodies: antibody quality metrics
  • Sequencing: sequencing quality metrics

Each metric per section has associated an info button and by overlying your mouse pointer over, a pop-up will show a message with the description of the metric:

QC Report metric info button

When one of the metrics show an unexpected behavior, it will show a small warning icon. By overlying the mouse pointer on that icon, a message pop-up on the warning will appear.

QC Report metric warnings

Parameters

QC Report parameters section

The parameters section shows the specific parameters that have been input to each stage in the nf-core/pixelator pipeline run.

You can also activate the "Show all parameters" button that will show all the parameters that are input to each stage. When all parameters are displayed, the parameters that the user has input are marked with a pencil icon.

QC Report all parameters section

Cells

The cells section show the summary of the sample and MPX assay metrics after calling for cells.

The default view will show a "Cell calling" dashboard where you can see the distribution of cells called (colored) as a scatter plot of the distribution of their size in edges by the number of unique antibody markers detected on them. The non-cell components are greyed out in the background of the distribution.

The residuals of the distribution are zoomed in as the cell distribution is so much smaller as compared to the background component distribution.

Each axis values can be displayed in a linear or logarithmic (log10, default) scale. And you can switch between them.

QC Report cells distribution section

A second dashboard called "Component size distribution" shows the edge rank plot that corresponds as to where the threshold for cell calling was found.

In this view you can also display linear or logarithmic (default) scales.

QC Report cells edgerank section

There is a collapsed button ("View Dimensionality Reduction Projection of Cell") that contains two dashboards to understand better the content of your sample. The left UMAP projection shows an UMAP projection with the total edge antibody counts per cell as background color scale. The right UMAP projection shows the clusters created after leiden clustering. When pixelator process each sample on the annotation stage, UMAPs are created with the default scanpy parameters. Those clusters show groups of cells with similarities in terms of antibodies presence.

QC Report cells dimensionality reduction section

Cell Annotations

If the optional annotation step into major groups of PBMCs has been activated in pixelator, the cell annotation section includes a table with either the percentages or total values of major PBMC cell types.

Currently followed by two UMAP projections to browse cell group annotations (if available) and another for counts per antibody. Only one antibody at a time can be selected. Both panels present raw molecule antibody counts.

QC Report cell annotation section umap

On the left dashboard, different major groups of PBMCs can be selected and their number of antibodies per cell retrieved. But if the annotation is not available, these panels will appear greyed out and all the cells are assigned to the "Unknown cells" group.

QC Report cell annotation section pbmc

On the right dashboard, you can search for markers of interest, one at a time. There is also search functionality that allows you to search between the antibodies in the panel list.

QC Report cell annotation section markers

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The functionality across the two UMAP panels allow MPX customers to look for antibody specificity and validation in annotated clusters of cells.

The last dashboard panel in this section represents count information per antibody in violin plots. However, this information comes in a log10 scale for better comparison. The information can be sliced across major groups of PBMCs when annotated.

QC Report cell annotation section markers

Also, several antibodies can be further selected in the "filter antibodies" menu. An active selection is saved and users can deselect those antibodies if not important anymore. This functionality is an useful to study antibodies with known cell type annotations and their level of signal and background in the sample (e.g. CD19 and CD20 for B-cells). Plots in dashboards are interactive and in the case of violin plots, several summary metrics can be extracted.

QC Report cell annotation section markers selection

Antibodies

The antibodies section describes several of the metrics of reads containing antibody information either if in finally called cells or just components.

QC Report antibodies section in percentage

The user can toggle between percentages and absolute values of the metrics with a toggle.

QC Report antibodies section in values

If the user expands the "View Barplots of Antibodies", the antibody section displays a dashboard with percentages of the number of antibody reads decoded and assigned to each antibody in percentages.

QC Report antibodies section barplot

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At the moment, pixelator doesn't compute a distribution of the antibodies per cell by different numbers of subsampled reads.

Sequencing

The sequencing section is showing different metrics that allows troubleshooting if parts of the amplicon sequenced have been subjected to any bias.

QC Report sequencing section

info

At the moment, pixelator doesn't compute a display of the saturation curve in the sequencing section.