Skip to content

Sample-sheet and barcodes

See example sample-sheet.

sci-rocket requires a sample sheet (.tsv) with at least the following required columns.

Either one or both of the following columns:

  • path_bcl: Path to folder containing the BCL files.
  • path_fastq: Path to folder containing the Undetermined_S0_R1_001.fastq.gz and Undetermined_S0_R2_001.fastq.gz files (if bcl2fastq has already been run).

Additional required columns:

  • experiment_name: Experiment name (e.g., experimentXYZ), used to associate all downstream files and underlying samples.
  • p5: PCR (p5) index(es) (e.g. A01:H01, or column(s) of a 96-well index plate) used to identify the sample during demultiplexing.
  • p7: PCR (p7) index(es) (e.g. G01:G12, or rows(s) of a 96-well index plate) used to identify the sample during demultiplexing.
  • rt: RT barcode(s) (e.g. P01-A01:P01-A12) used to identify the sample during demultiplexing.
  • sample_name: Name of the demultiplexed sample, used to generate sample-specific files.
  • species: Reference species (e.g. mouse or human).
  • n_expected_cells: Number of expected cells in the (demultiplexed) sample (used during UMI filtering).
  • p5 and p7 are used to denote the PCR indexes belonging to a particular sample / cell. The indexes are translated to all relevant combinations within the sequencing-run using the 96-well index plate layout of 8x rows (A:H) and 12 columns (01:12). To specify one or multiple p5/p7 ranges, use the following format:
  • p5 (1 column): A01:H01
  • p5 (1.5 columns): A01:H01,A02:D02
  • p5 (2 columns): A01:H01,A02:H02
  • p7 (1 row): G01:G12
  • p7 (1.5 rows): G01:G12,H01:H06
  • p7 (2 rows): G01:G12,H01:H12
  • The rt is used to denote the RT barcode belonging to a particular sample / cell. The indexes are translated to all relevant combinations within the sequencing-run. To specify one or multiple RT strips, use the following format:
  • One RT: P01-A01
  • Multiple RT (1 strip): P01-A01:P01-A12
  • Multiple RT (Multiple strips): P01-A01:P01-H03; This will include all RT barcodes from P01-A01 to P01-H03.

  • Multiple RT (Multiple strips + plates): P01-A01:P01-D12,P02-A01:P02-B12; This will include all RT barcodes from P01-A01 to P01-D12 and P02-A01 to P02-B12.

  • species should be present in the config.yaml file with their respective genome sequences (.fa) and gene-annotations (.gtf) used to generate mapping indexes.

Barcode design

The workflow requires a file (.tsv) containing the barcodes used in the experiment with at least the following required columns:

  • type: Type of barcode (ligation, p5, p7 or rt).
  • barcode: Name of the barcode (e.g. A01).
  • sequence: Nucleotide sequence of the barcode.

Hashing sheet

The hashing workflow requires a separate file (.tsv) containing the hashing schematics used in the sample with at least the following required columns:

  • hash_name: Name of the hashing experiment (e.g. hash_exp1).
  • barcode: Sequence of the respective hashing barcode (e.g. GGTTGGCGAC).

To specify which samples are to be hashed (and using which hashing-sheet), add an additional column (hashing) in the sample-sheet to each each sample with the respective path to the hashing sheet.

Haplotyping (optional; Mus musculus cross-experiments only)

As optional procedure, sci-rocket can be used to further haplotype the sex-chromosome X of the demultiplexed samples, e.g. in the case of mouse F1 cross-hybrids. For this, the following columns can be added to the sample-sheet:

  • strain1: Name of the first strain (e.g. B6 (C57BL/6J)).
  • strain2: Name of the second strain (e.g. CAST/EiJ).

These strains should be present in the Mouse Genome Project (MGP) database. For C57BL/6J (wt), use B6 as strain name.

This will add haplotype-specific read tags (HP) to the STARSolo BAM files and will output an additional haplotyping folder which contains cell-based read-counts per gene per haplotype (H1, H2, UA) for chromosome X.