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Extended Throughput Kit v1.1

When using additional final distribution (PCR) plates from the Extended Throughput Kit v1.1, each plate has the same set of 96 i5-index sequences, but receives a different set of i7 index sequences from the P7 primer pool used (RNA-A-AP1 to RNA-A-AP4). This data is analyzed by generating separate "fastq libraries" for each plate (i7 pool), analyzing each separately through the core workflow (barcodeParser, STARSolo, etc.) and then combining the outputs at the single-cell level (gene expression matrix and metrics).

Cells from different plates are distinguished by appending the library-name (P7 primer pool) to the cell barcode in the gene expression matrix outputs.

Single Sequencing Run

If all plates are sequenced together in the same run, this can be done in one nextflow analysis run:

  • Create a samples.csv for all plates and samples
    • List each sample on each plate (repeating the sample name for each plate used)
    • Set the libIndex column to the name of the barcode pool (e.g. RNA-A-AP1)
    • See samples.ext.csv for an example with two samples on two plates
  • Launch the workflow
    • This will produce outputs for each individual plate as well as merged outputs (with default merge set), combining all cells for each sample across all plates

A small test-case can be run with:
nextflow run /PATH/TO/ScaleRna -profile PROFILE -params-file /PATH/TO/ScaleRna/docs/examples/extended-throughput/runParams.yml --outDir output.ext

Merging Across Multiple Runs

If plates were sequenced on separate sequencing runs, each plate can first be analyzed individually and the results later merged in a separate nextflow run

  • Create a samples.csv for each separate plate
    • Including the libIndex column with the P7 pool used for that plate
  • Run the nextflow analysis workflow separately for each plate
  • Create another samples.csv that lists all samples and plates to be merged
    • Add a new resultDir column that points to the workflow output directories for the individual plate runs
    • See samples.ext-merge.csv
  • Run the workflow with the --reporting option
    • --reporting uses the previous alignment results instead of re-processing all reads
    • Do not specify any input reads at this step (no --runFolder or --fastqDir)

Fastq Generation

To run this workflow starting from fastq files, rather than a runFolder / BCLs, see Fastq Generation