Sample-sheet and barcodes

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 row): P01-A01:P01-A12

Multiple RT (1 column): P01-A01:P01-H01

Multiple RT (2 columns): P01-A01:P01-H02

Multiple RT (rectangular region): P01-B02:P01-E04; This will include all RT barcodes from the rectangle with P01-B02 at the top left and P01-E04 at the bottom right:

P01 1 2 3 4 5

A . . . . .

B . X X X .

C . X X X .

D . X X X .

E . X X X .

F . . . . .

Multiple RT (multiple plates): P01-B02:P01-E04,P02-A01:P02-A12; This will include the same RT barcodes from P01 as the previous example, plus row A from P02.

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.