Cell Ranger - ATAC

What is Cell Ranger?

Cell Ranger is a set of analysis pipelines that process Chromium single cell data to align reads, generate feature-barcode matrices and perform clustering and gene expression analysis for 10X Genomics Chromium Technology[1].

Cell Ranger - ATAC in Partek Flow

The 'cellranger-atac count' pipeline from Cell Ranger ATAC v2.0[2] has been wrapped in Partek^® Flow^® as Cell Ranger - ATAC task. It takes FASTQ files from 'cellranger-atac mkfastq' and performs ATAC analysis including reads filtering and alignment, barcode counting, identification of transposase cut sites, peak and cell calling, count matrix generation. Its outputs then becomes the starting point for downstream analysis for scATAC-seq data in Flow.

Running Cell Ranger - ATAC in Flow

To run the Cell Ranger - ATAC task for scATAC-seq data in Flow, select Unaligned reads datanode, then select Cell Ranger - ATAC in the 10x Genomics section (Figure 1).

Figure 1. Selecting the Cell Ranger - ATAC task for converting fastqs to Single cell counts.

Similar to Cell Ranger - Gene Expression task, the first time user will be asked to create a Reference assembly. In Partek^® Flow^®, we will use Cell Ranger ARC 2.0.0 to create reference assembly for all 10x Genomics analysis pipelines. Please refer to our Cell Ranger - Gene Expression task manual on how to build or use Reference assembly.

Once the right assembly has been chosen/provided, simply press the Finish button to run the task with default settings. The reference assembly of ‘Homo sapiens (human) - hg38’ has been used as an example here (Figure 2).

Figure 2. Run Cell Ranger - ATAC task in Flow.

A new data node named Single cell counts will be displayed in Flow if the task has been finished successfully (Figure 3). This data node contains a filtered peak barcode count matrix. To open the task report when the task is finished, double click the output data node, or select the Task report in the Task results section after single clicking the data node. Users then will find the task report (Figure 4) is the same to the ‘Summary HTML’ from Cell Ranger ATAC output.

Figure 3. The finished Cell Ranger - ATAC task in Flow.

Cell Ranger - ATAC task report in Flow

Task report is sample based. Users can use the dropdown list on the top left to switch samples. Under the sample name, there are two tabs on each report - Summary report and Data Quality report (Figure 4). Important information on Estimated Number of Cells, Median high-quality fragments per cell, Fraction of high-quality fragments overlapping peaks, as well as information on Sample, Sequencing, Cells and Cell Clustering are summarized in different panels. The Barcode Rank plot and Fragment Distribution plot have also been included as an important piece in the Cells panel in the Summary report (Figure 4). Descriptions of metrics in the following sections can also be found by clicking the to the section header in the Summary HTML file itself.

Figure 4. The example report of Cell Ranger - ATAC task in Flow.

The Library Complexity section in Data Quality report plots the observed per cell complexity, measured as median unique fragments per cell, as a function of mean reads per cell. (Figure 5). WhileIn the Mapping section displays the Insert Size Distribution plot, and metrics derived from it. Single Cell ATAC read pairs produce detailed information about nucleosome packing and positioning. The fragment length distribution captures the nucleosome positioning periodicity. The Targeting section shows profiling of the chromatin accessibility behavior of the library at epigenetically relevant regions in the genome. The Enrichment around TSS plot is helpful to assess the signal-to-noise ratio of the library. It is well known that TSSs and the promoter regions around them have a higher degree of chromatin accessibility compared to the other regions of the genome. The Peaks targeting plot presents the variation in the number of on-target fragments, or fragments that overlap peaks, within each barcode group. A higher percentage of the barcode fragments overlap peaks is expected for cell-associated barcodes.

Figure 5. Data Quality report of Cell Ranger - ATAC task in Flow.

Other parameter to adjust (Figure 2):

Subsample percentile: Downsample to preserve this fraction of reads

Users can click Configure to change the default settings in Advanced options (Figure 2).

Override peak caller: To override the peak caller, users specify peaks to use in downstream analyses from supplied 3-column BED file. The supplied peaks file must be sorted by position and not contain overlapping peaks; comment lines beginning with `#` are allowed.

Force cells: Define the top N barcodes with the most fragments overlapping peaks as cells and override the cell calling algorithm. N must be a positive integer <= 20,000. Use this option if the number of cells estimated by Cell Ranger -ATAC is not consistent with the barcode rank plot.

Memory limit (GB): Restricts Cell Ranger - ATAC to use specified amount of memory (in GB) to execute pipeline stages.

Partek Flow Documentation

Page tree

What is Cell Ranger?

Cell Ranger - ATAC in Partek Flow

Running Cell Ranger - ATAC in Flow

Cell Ranger - ATAC task report in Flow

References