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Once the Genomics production has been picked, users will be asked to create a Reference assembly if it is the first time to run the Cell Ranger - Gene Expression task (Figure 2). In Partek Flow, we will use Cell Ranger ARC 2.0.0 to create reference assembly for all 10x Genomics analysis pipelines. To create and use a reference assembly, Cell Ranger ARC requires a reference genome sequence (FASTA file) and gene annotations (GTF file).

Clicking the big grey button of Create Cell Ranger ARC 2.0.0 reference would pop up a new window where lists the requirements that users need to fill in (Figure 3). To create the same reference genomes (2020-A) that are provided in Cell Ranger by default, the transcriptome annotations are respectively GENCODE v32  for human and vM23 for mouse, which are equivalent to Ensembl 98[3]. If users don't have any options in the dropdown list, they can click Add annotation model (GTF file) for Index, or New assembly... (FASTA file)for Assembly and upload the files. 

Once the right options has been chosen/provided, simply press the Create button to finish. The reference assembly of ‘Homo sapiens (human) - hg38’ has been created as an example here (Figure 4).

The main task menu will be refreshed as above (Figure 4) for gene expression data if references have been added. Users can go ahead click the Finish button to run the task as default. 

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A new data node named Single cell counts will be displayed in Flow if the task has been finished successfully (Figure 6). This data node contains a filtered feature barcode count matrix for gene expression data, but a unified feature-barcode matrix that contains gene expression counts alongside Feature Barcode counts for each cell barcode for Feature Barcode data. 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 7) is the same to the ‘Summary HTML’ from Cell Ranger output.

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