Cell Ranger alternatives to generate gene-count matrices for 10X data

This count workflow generates gene-count matrices from 10X FASTQ data using alternative methods other than Cell Ranger. If your data start from BCL files, please first run BCL Convert to demultiplex flowcells to generate FASTQ files.

Prepare input data and import workflow

1. Import `count`

Import count workflow to your workspace.

See the Terra documentation for adding a workflow. The count workflow is under Broad Methods Repository with name “cumulus/count”.

Moreover, in the workflow page, click the Export to Workspace... button, and select the workspace to which you want to export count workflow in the drop-down menu.

2. Prepare a sample sheet

2.1 Sample sheet format:

The sample sheet for count workflow should be in TSV format, i.e. columns are seperated by tabs not commas. Please note that the columns in the TSV can be in any order, but that the column names must match the recognized headings.

The sample sheet describes how to identify flowcells and generate channel-specific count matrices.

A brief description of the sample sheet format is listed below (required column headers are shown in bold).

Column

Description

Sample

Contains sample names. Each 10x channel should have a unique sample name.

Flowcells

Indicates the Google bucket URLs of folder(s) holding FASTQ files of this sample.

The sample sheet supports sequencing the same 10x channel across multiple flowcells. If a sample is sequenced across multiple flowcells, simply list all of its flowcells in a comma-seperated way. In the following example, we have 2 samples sequenced in two flowcells.

Example:
Sample  Flowcells
sample_1        gs://fc-e0000000-0000-0000-0000-000000000000/VK18WBC6Z4/sample_1_fastqs,gs://fc-e0000000-0000-0000-0000-000000000000/VK10WBC9Z2/sample_1_fastqs
sample_2        gs://fc-e0000000-0000-0000-0000-000000000000/VK18WBC6Z4/sample_2_fastqs
Moreover, if one flowcell of a sample contains multiple FASTQ files for each read, i.e. sequences from multiple lanes, you should keep your sample sheet as the same, and count workflow will automatically merge lanes altogether for the sample before performing counting.

2.2 Upload your sample sheet to the workspace bucket:

Use gsutil (you already have it if you’ve installed Google cloud SDK) in your unix terminal to upload your sample sheet to workspace bucket.

Example:
gsutil cp /foo/bar/projects/sample_sheet.tsv gs://fc-e0000000-0000-0000-0000-000000000000/

3. Launch analysis

In your workspace, open count in WORKFLOWS tab. Select the desired snapshot version (e.g. latest). Select Process single workflow from files as below

and click SAVE button. Select Use call caching and click INPUTS. Then fill in appropriate values in the Attribute column. Alternative, you can upload a JSON file to configure input by clicking Drag or click to upload json.

Once INPUTS are appropriated filled, click RUN ANALYSIS and then click LAUNCH.

Workflow inputs

Below are inputs for count workflow. Notice that required inputs are in bold.

Name	Description	Example	Default
input_tsv_file	Input TSV sample sheet describing metadata of each sample.	“gs://fc-e0000000-0000-0000-0000-000000000000/sample_sheet.tsv”
genome	Genome reference name. Current support: GRCh38, mm10.	“GRCh38”
chemistry	10X genomics’ chemistry name. Current support: “tenX_v3” (for V3 chemistry), “tenX_v2” (for V2 chemistry), “dropseq” (for Drop-Seq).	“tenX_v3”
output_directory	GS URL of output directory.	“gs://fc-e0000000-0000-0000-0000-000000000000/count_result”
run_count	If you want to run count tools to generate gene-count matrices.	true	true
count_tool	Count tool to generate result. Options: “StarSolo”: Use STARsolo. “Optimus”: Use Optimus pipeline, developed by the Data Coordination Platform team of the Human Cell Atlas. “Bustools”: Use Kallisto BUSTools. “Alevin”: Use Salmon Alevin.	“StarSolo”	“StarSolo”
docker_registry	Docker registry to use. Notice that docker image for Bustools is seperate. “quay.io/cumulus” for images on Red Hat registry; “cumulusprod” for backup images on Docker Hub.	“quay.io/cumulus”	“quay.io/cumulus”
config_version	Version of config docker image to use. This docker is used for parsing the input sample sheet for downstream execution. Available options: `0.2`, `0.1`.	“0.2”	“0.2”
zones	Google cloud zones to consider for execution.	“us-east1-d us-west1-a us-west1-b”	“us-central1-a us-central1-b us-central1-c us-central1-f us-east1-b us-east1-c us-east1-d us-west1-a us-west1-b us-west1-c”
num_cpu	Number of CPUs to request for count per channel. Notice that when use Optimus for count, this input only affects steps of copying files. Optimus uses CPUs due to its own strategy.	32	32
disk_space	Disk space in GB needed for count per channel. Notice that when use Optimus for count, this input only affects steps of copying files. Optimus uses disk space due to its own strategy.	500	500
memory	Memory size in GB needed for count per channel. Notice that when use Optimus for count, this input only affects steps of copying files. Optimus uses memory size due to its own strategy.	120	120
preemptible	Number of maximum preemptible tries allowed. Notice that when use Optimus for count, this input only affects steps of copying files. Optimus uses preemptible tries due to its own strategy.	2	2
merge_fastq_memory	Memory size in GB needed for merge fastq per channel.	32	32
starsolo_star_version	STAR version to use. Currently only support “2.7.3a”. This input only works when setting count_tool to `StarSolo`.	“2.7.3a”	“2.7.3a”
alevin_version	Salmon version to use. Currently only support “1.1”. This input only works when setting count_tool to `Alevin`.	“1.1”	“1.1”
bustools_output_loom	If BUSTools generates gene-count matrices in `loom` format. This input only works when setting count_tool to `Bustools`.	false	false
bustools_output_h5ad	If BUSTools generates gene-count matrices in `h5ad` format. This input only works when setting count_tool to `Bustools`.	false	false
bustools_docker	Docker image used for Kallisto BUSTools count. This input only works when setting count_tool to `Bustools`.	“shaleklab/kallisto-bustools”	“shaleklab/kallisto-bustools”
bustools_version	kb version to use. Currently only support “0.24.4”. This input only works when setting count_tool to `Bustools`.	“0.24.4”	“0.24.4”
optimus_output_loom	If Optimus generates gene-count matrices in `loom` format. This input only works when setting count_tool to `Optimus`.	true	true

Workflow outputs

See the table below for count workflow outputs.

Name	Type	Description
output_folder	String	Google Bucket URL of output directory. Within it, each folder is for one sample in the input sample sheet.