What are the steps for preprocessing single cell data generated from BD Rhapsody system ?

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If there is a tutorial for the same please share them.

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Welcome @abz-xyz

The tutorials below for ACAT analysis are likely where to start. But let’s bring is some scientists from our single cell community to confirm. Hi @pavanvidem – would you be able to help or suggest someone who can? I also cross posted over to the Single Cell chat group. Feel free to join the chat! → You're invited to talk on Matrix

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hi @abz-xyz,
I see the following three possibilities on Galaxy.

  1. UMI-tools:
    • Use UMI-tools extract tool with Barcode Extraction Method as Regex to extract barcodes and UMIs. There are some guidelines on what regex to use for BD-Rhapsody: FAQ — UMI-tools documentation
    • Once you have it, you can use UMI-tools count to generate a count matrix.
  2. STARSolo:
    • Download the barcode whitelists (CLS1, CLS2 and CLS3) from here: BD Rhapsody WTA. Also please check whether the library layout is the same as yours. Then follow this and this issues from STARSolo to check what parameters to set. The main parameters to set are:
      • Type of single-cell RNA-seq as inDrop
      • To generate --soloCBposition 0_0_0_8 0_21_0_29 0_43_0_51 (as shown in the issue), repeat Cell barcode whitelist information parameter 3 times.
        • Select the downloaded CLS1.txt
        • Input Read start, 0, Read start, 8 for the next 4 parameters.
        • Select the downloaded CLS2.txt
        • Input Read start, 21, Read start, 29
        • Select the downloaded CLS3.txt
        • Input Read start, 43, Read start, 51
      • To generate --soloUMIposition 0_52_0_59, input the following params:
        • Start anchor base for UMI: Read start
        • 0-based position of the UMI start with respect to the anchor base: 52
        • End anchor base for UMI: Read Start
        • 0-based position of the UMI end with respect to the anchor base: 59
  3. If you are analyzing any public datasets, please start from the count data directly. Normally, they provide the count matrices or Anndata objects. Import Anndata tool allows importing from various data formats, like UMI-tools count matrix or MatrixMarket format.

The main problem with the STARSolo approach is that Galaxy currently supports only 2 barcode whitelists input with the inDrop option. We can change it to take 3 inputs, if it is really needed. But I suggest trying option 3 first and then option 1 before coming to option 2.

All the best,
Pavan

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