Specifying Inputs

Samplesheet

The samplesheet is a file in CSV or TSV format that specifies the input data for the nf-core/pixelator pipeline.

The samplesheet is a required input for the pipeline and is used to configure the pipeline run. It allows you to specify multiple samples and their associated data in a single file, making it easier to manage and run the pipeline on multiple samples at once.

You can include any information you want in the samplesheet; however, it must contain the following columns with information required for running pixelator:

• Sample names in the sample column.
• Sample aliases in the sample_alias column. This is the alias for the sample; a short, unique identifier for the sample that is used in the Proxiome Experiment Summary report. If not provided, sample will be used.
• A condition for each sample, e.g., control/treatment, in the condition column.
• The assay design in the design column.
• The panel used, either as the panel name in a panel column or as the path to a panel CSV file in a panel_file column, depending on the scenario.
• The paths to the FASTQ files in the fastq_1 column (and fastq_2 if present).

For Proxiome v2, the samplesheet also requires the pool and hash_index columns.
You will find examples and more detailed information about how to set up the samplesheet here.

Design

The assay design specifies the expected molecular layout of the sequencing libraries and is needed for pixelator to identify the correct protein ID and unique molecular identifier regions within each read. Which design to use is specified in the samplesheet.

Use one of the two designs provided in pixelator:

• proxiome-v1 for samples ran with Proxiome v1 kits.
• proxiome-v2 for samples ran with Proxiome v2 kits supporting sample hashing.

See the nf-core-pixelator documentation for more details.

Panel

The panel file specifies which antibody targets (markers) are included in the assay and which sequences they are associated with. The file is a CSV with a YAML-formatted metadata header at the top.

Panel files can be specified in two ways:

• Using a predefined panel name to use the default built-in panels.
• Passing a CSV file with a customized panel.

For most cases, use one of the panel files provided in pixelator.
See Choosing panel file for data processing for a table of the currently available panels and which kit lot they correspond to.

Creating custom panels

Follow these instructions to add your own custom markers to a released panel.
Find the appropriate base panel file in the pixelator repository and make a copy of it.
Using your favorite editor, do the following modifications to the panel file:

• Add a new row at the bottom of the file.
• Choose an ID or name for your marker and put it in the marker_id column.
• Copy the appropriate antibody barcode sequence from the table below to the sequence_1 and sequence_2 columns. The same sequence should go into both columns.
• Leave the control and sample_hashing columns empty if they are present.
• Optionally, for any additional columns present, e.g., full_name, fill in appropriate values.

If desired, repeat these steps for up to two out of the four antibody barcodes reserved for custom markers.
The allowed combination of barcodes depend on the kit used (see the Custom conjugation kit to barcode pair mapping table below for details).

To also add on CAR T Barcoded Antibodies, follow the above steps for up to two of the markers defined in table Antibody barcodes for CAR T Barcoded Antibodies

Note: It is strongly recommended to change the name field in the header, e.g., to "my-custom-PNA-panel", to avoid confusion with the original panel.

Finally, to use your custom panel csv file, you will need to add it to the sample sheet using the panel_file column, this column allows for specifying panel using the path to the panel csv rather than a panel name from the predefined set.

Antibody barcodes reserved for custom markers:

Name	Sequence
BC 1	TAACGAATCC
BC 2	CAGTCCGATG
BC 3	GTTGCCTGAC
BC 4	CAATGTAACC

Custom conjugation kit to barcode pair mapping

Kit	Barcodes
PROXCUST001	BC3 + BC4
PROXCUST002	BC1 + BC2
PROXCUST003	BC2 + BC4

Antibody barcodes for CAR T Barcoded Antibodies:

Name	Sequence
FLAG	CTTAACATCT
FMC63	CGATTGGCAC
G4S	TTCTTTGATG

Remember, when editing panel files, always adhere to the file format specification in the expandable section below.

Panel File Format

The panel file consists of two parts:

1. YAML Metadata Header (at the top, each line prefixed with # ):

# name: <panel_name>
# version: <semantic_version>
# product: <product_name>         # (optional)
# description: <description>     # (optional)
# aliases: [<alias1>, <alias2>]  # (optional)
# archived: false                # (optional)

Metadata fields:

• name Name of the panel (string, required)
• version Semantic version string (string, required)
• product Product identifier (string, optional)
• description Description of the panel (string, optional)
• aliases List of alternative names (array of strings, optional)
• archived Whether the panel is archived (boolean, optional)

2. CSV Table (after the YAML header):

Required columns for the panel file:

• marker_id Unique identifier for the marker (must be unique, no underscores or spaces)
• control Whether the marker is a control "yes" (all other values are considered as specifying a non control marker)
• sequence_1 First PID DNA sequence for the marker (must be unique, only A/T/C/G characters)
• sequence_2 Second PID DNA sequence for the marker (must be unique, only A/T/C/G characters)

To run a v2 assay with sample hashing, the panel file also needs to include:

• sample_hashing column with the value "yes" for the markers to be used for sample hashing (all other values are considered as specifying a non-hashing marker).

All other non-required columns that might be present in the panel file, e.g., full_name, alt_id, and/or uniprot_id, are passed on to the var table of the AnnData object included in the final pixelfile.

Notes:

• The YAML header must come first.
• The CSV table must immediately follow the header (no blank lines).

For more details about the panel file and format, see the source code in the pixelator repository.

Example panel file

# name: Example PNA Antibody Panel with custom markers
# version: 1.0.0
# product: Example Product
# description: Example PNA antibody panel for demonstration
marker_id,control,sequence_1,sequence_2,sample_hashing
CD3,no,ATCGATCGAT,CGTACGTACG,
CD19,no,GCTAGCTAGC,TAGCTAGCTA,
mIgG1,yes,ATGCTAGTCG,ATGCTAGTCG,
B2M-1,,TGAGTGGTGA,TGAGTGGTGA,yes
B2M-2,,TAGAGGAGGC,TAGAGGAGGC,yes
custom-marker-1,,TAACGAATCC,TAACGAATCC,
custom-marker-2,,CAGTCCGATG,CAGTCCGATG,
custom-marker-3,,GTTGCCTGAC,GTTGCCTGAC,
custom-marker-4,,CAATGTAACC,CAATGTAACC,