Split Read Mapping by Cell

Split Read Mapping by Cell splits an input Read Mapping (Image read_track_16_n_p) according to groupings provided by Cell Clusters (Image cell_clusters_16_n_p) or Cell Annotations (Image cell_annotations_16_n_p). It can be found in the Toolbox here:

        Toolbox | Single Cell Analysis (Image sc_folder_closed_16_n_p) | Chromatin Accessibility (Image sc_atac_seq_folder_open_16_n_p) | Split Read Mapping by Cell (Image split_reads_cell_16_n_p)

There are two types of output:

The options control the groups of cells for which an output is produced:

The tool also outputs a Report (Image proteinreport) summarizing the input and the resulting cell groups.

Peak graph tracks

The Create peak graph tracks option creates a graph of fragment coverage for each group of cells. Only paired end reads are used to create the graph - broken pairs are discarded. Fragments are corrected to the cut site by offsetting read start sites by +4nt for forward reads and -5nt for reverse reads. The peak graph track often provides a more intuitive visualization of peaks than a Read Mapping and uses much less diskspace. The visualization is more intuitive because the unsequenced part of each fragment that lies between the two reads of a pair is counted towards the coverage of the peak graph, but does not count towards the coverage of the Read Mapping.

It is recommended to only create peak graph tracks on read mappings that have been produced by Single Cell ATAC-Seq Analysis, as otherwise the presence of duplicate reads can make peaks less clear.

Peak graph tracks can be scaled in two ways. Scaling does not affect the relative height of peaks within the same track, and so is only useful when comparing peaks in two different tracks:

To visualize the effect of scaling in a Track List, all graph tracks must be shown on the same scale. To do this, check the Fix graph bounds option in the Side Panel. The effect of different settings is shown in figures 12.4-12.6.

Image split_mapping_autoscale
Figure 12.4: A Track List showing the Read Mapping coverage graph (top), called peaks, and peak graph tracks for three groups of cells of very different sizes. Fix graph bounds is not checked in the Side Panel, so each graph track is independently rescaled to use the available space. This means that the graph tracks for each group appear the same regardless of whether they have no scaling or are scaled by number of cells. Data is for one sample from [Taavitsainen et al., 2021].

Image split_mapping_no_scale
Figure 12.5: The same Track List as in figure 12.4, but only showing the graph tracks without scaling and with Fix graph bounds checked in the Side Panel. There are many more cells in group 3 than in group 1, and this is reflected by the heights of the graphs - the signal at each of the two peaks is much stronger in group 3 than in group 1.

Image split_mapping_by_group_size
Figure 12.6: The same Track List as in figure 12.4, but only showing the graph tracks with scaling and with Fix graph bounds checked in the Side Panel. The heights of the graphs are much greater in group 1 than in group 3. This is because a greater fraction of the cells in group 1 than in group 3 have reads in the peaks.

Reads tracks

The Create reads tracks option creates a Read Mapping for each group of cells. Unlike Create peak graph tracks, no filtering or post-processing of the reads is applied: the output includes paired end reads and broken pairs, and the original alignment coordinates are preserved (i.e. there is no correction to the cut site).

Report

The report lists how many fragments and cells were found in the input Read Mapping:

For each resulting cell group, the number of cells in the group is reported.