Omnition Analysis Software

Omnition Analysis Software: Understanding the ATAC Report

The Omnition Analysis Software outputs a single HTML report containing all samples that are processed in a run of the pipeline. This
guide explains metrics for each plot and table reported by Omnition. Tooltips are also accessible by hovering over thenext to theheaders of plots and tables.

Overview of Report Tabs
The report includes 6 sections:• Experiment Summary• Read QC• Analysis Statistics• Deconvolution• ATAC Metrics• Results
Follow the tabs on the top of the report page to view each section for a given sample. Switch between samples via the sample ID
dropdown list found in the following sample-level sections: Analysis Statistics, Deconvolution, ATAC Metrics, and Results.

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Experiment Summary
Summary Table

Messages

The Experiment Summary table provides an at-a-glancesummary of key metrics and statistics for each sample
analyzed in the same pipeline run.

The Messages section shows status messages output byOmnition.

Total Cells Recovered:Calculated cell count after quality control and deconvolution.
Mean Valid Read Pairs per Cell:

Average number of read pairs containing a complete barcode
sequence for each called cell.
Median Unique Nuclear Fragments per Cell:

Median number of unique fragments that aligned to nuclear
DNA in a cell.
Median Estimated Library Size per Cell:

Median predicted number of unique nuclear fragments percell in the sequencing library.
Aligned Read Pairs (%):

Percentage of read pairs that aligned to the genome andmitochondrial DNA.
Aligned Duplicate Read Pairs (%):

Percentage of proper read pairs with similar sequences andalignment positions.
Aligned Mitochondrial Read Pairs (%):

Percentage of proper read pairs that aligned to mitochondrial
DNA.
Transcription Start Site (TSS) Enrichment Score:

The average ratio of coverage at the TSS normalized tocoverage at a set distance from the TSS.
Peaks Called:

The number of unique accessible chromatin regions identified
in the sample.
Fraction of Reads in Peaks (FRiP):

The fraction of unique reads that aligned to identified
accessible chromatin regions (peaks).

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Read QC
Quality Score Plot

Sequence Trace Heat Map

The Quality Score plot shows the mean Phred quality scoreat each base position as an indication of the base callaccuracy.

The Sequence Trace heat map shows the proportion of eachof the four DNA bases being called at each base position.

Sequence Trace Plot
Click each tile in the heat map to switch to the trace plot.
Click “Return to Heat Map” (circled) to return.

The Sequence Trace plot shows the mean nucleotide
composition at each base position.

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Analysis Statistics
Alignment Metrics Table

Pipeline Summary Table

Input Read Pairs:

Number of read pairs input into alignment.

Pipeline Summary table shows the proportion of read pairs
retained throughout the analysis.

Aligned Read Pairs:

Read Pairs with Valid Barcodes:

Percentage of read pairs that aligned to the genome andmitochondrial DNA.

Read pairs containing complete barcode sequences.
High-Quality Read Pairs:

Aligned Proper Read Pairs:

The percentage of deduplicated proper read pairs that met
the quality threshold and insert size requirements.

Percentage of read pairs that align to the genome andmitochondrial DNA in the correct orientation within areasonable distance of each other.

High-Quality Pairs Remaining after Blocklist Removal:

The percentage of deduplicated proper read pairs alignedwith high-quality in regions of interest.

Aligned Duplicate Read Pairs:

Percentage of proper read pairs with similar sequences andalignment positions.

Deduplicated Above-Knee Read Pairs:

The percentage of unique high-quality read pairs in regions ofinterest associated with called cells.

Aligned Mitochondrial Read Pairs:Percentage of proper read pairs that aligned to mitochondrialDNA.

Read Pairs per Index Table
For single cell combinatorial indexing ATAC data with theTagmentation Index (TI), the Read Pairs per Index table
reports the proportion of read pairs with valid barcodes
attributed to each unique combination of FASTQ and TI in asample.

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Alignment Distribution PlotSequence Saturation Plot

The Alignment Distribution plot shows chromosome locationsof aligned and unaligned reads.

The Sequence Saturation plot shows the number of uniquefragments identified per cell compared to the number of valid
reads in a sample, as a function of sequencing saturation.

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Deconvolution
Bead Filtration Table

Bead Filtration Plot

The Bead Filtration table displays a quantification of what
happened in the bead filtration step.

The Bead Filtration plot shows the distribution of uniquenuclear fragments across individual bead barcodes. Thesignal below the inflection point is assumed to be noise.

Total Barcodes Observed:

The number of bead barcodes identified.
Barcodes Above Knee:

The number of bead barcodes associated with uniquenuclear fragments that is greater than the unique nuclear
fragment threshold at the knee.
Unique Nuclear Fragment Threshold at Knee:

The minimum number of unique nuclear fragments a beadbarcode must be associated with to be considered true signal.
Median Unique Nuclear Fragments per Bead Above the Knee:

The median number of unique nuclear fragments per beadbarcode that is greater than the threshold at the knee.
Total Cells Recovered:Calculated cell count after quality control and deconvolution.
Mean Valid Read Pairs per Cell:

Average number of read pairs containing a complete barcode
sequence for each called cell.
Median Unique Nuclear Fragments per Cell:

Median number of unique fragments that align to nuclear
DNA in a cell.
Median Estimated Library Size per Cell:

Median predicted number of unique nuclear fragments percell in the sequencing library, calculated using number ofunique nuclear fragments and duplication rate.

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Jaccard Index Plot

Beads per Partition PlotThe Jaccard Index plot shows the distribution of Jaccard
similarity index scores across bead barcode pairs. Pairs
with scores above the inflection point are assumed to beoriginating in the same partition.

The Beads per Partition plot shows the frequency of different
numbers of bead multiplets merged during deconvolution.

RETURNATAC Metrics
ATAC-Seq Metrics Table
Transcription Start Site (TSS) Enrichment Score:

Fraction of reads in peaks (FRiP):The average ratio of coverage at the TSS normalized tocoverage at a set distance from the TSS.

The fraction of unique reads that aligned to identified
accessible chromatin regions (peaks).

Total TSS Reads:

TSS Read Percentage:

The number of proper read pairs that aligned to a TSS.

The percentage of proper read pairs that aligned to a TSS.

Peaks Called:

The number of unique accessible chromatin regions identified
in the sample.

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Omnition Analysis Software: Understanding the ATAC ReportTSS Enrichment Plot

Insert Size Distribution Plot

The distribution of peaks relative to known transcription startsites (TSS).

The distribution of sequenced fragment sizes, indicative ofnucleosome positioning periodicity.

Unique Nuclear Fragments Plot

Unique Mitochondrial Fragments Plot

The distribution of unique nuclear fragments per cell.

The distribution of unique mitochondrial fragments per cell.

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Omnition Analysis Software: Understanding the ATAC ReportTSS Proportion Plot

Duplicate Proportion Plot

The aggregate distribution of proper read pairs aligned to aTSS per cell.

The distribution of aligned duplicate read pairs per cell.

Estimated Library Size Plot

Unique Fragments per Cell Plot

Predicted number of unique nuclear fragments per cell inthe sequencing library, calculated using number of uniquenuclear fragments and duplication rate.

The distribution of unique nuclear fragments across cells
identified after merging of above-knee bead barcodes.

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Results
Highlights

Crosstalk Statistics

Total Cells Recovered:

Total Cells Recovered:Calculated cell count after quality control and deconvolution.

Calculated cell count after quality control and deconvolution.

Median Unique Nuclear Fragments per cell:[Genome1] Cells:

Median number of unique fragments that align to nuclear
DNA in a cell.

The number of cell barcodes that have ≥90% (default) ofproper read pairs from [genome1].

Transcription Start Site (TSS) Enrichment Score:[Genome2] Cells:

The average ratio of coverage at the TSS normalized tocoverage at a set distance from the TSS.

The number of cell barcodes that have ≥90% (default) ofproper read pairs from [genome2].

Fraction of Reads in Peaks (FRiP):Mixed Cells:

The fraction of unique reads that aligned to identified
accessible chromatin regions (peaks).

The number of cell barcodes that have <90% (default) ofproper read pairs from a single species.
Observed Mixed-Species Multiplets:

The percentage of cell barcodes that have <90% (default)of proper read pairs from a single species quantifying
detectable multiplets.
Estimated Total Multiplets:

The percentage of the estimated total multiplets, including
same-species multiplets, calculated by doubling thepercentage of mixed-species multiplets.
Cell Purity:

The percentage of proper read pairs that are correctly
assigned with the cell barcodes’ species out of the total
proper read pairs.

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Crosstalk Density Plot

UMAP Cluster Plot

The Crosstalk Density plot shows the number of uniquenuclear fragments from each cell barcode aligned to aparticular species. Mixed cells are those with unique nuclear
fragments aligned to both species.

The UMAP Cluster plot displays Identification of clusters
based on differential chromatin accessibility across thegenome.

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