Getting Started =============== Running the alevin-fry pipeline ------------------------------- First, we need to generate a RAD file using alevin. The RAD file is created by mapping the sequencing reads against an index of the reference. We recommend using a `splici `_ reference index. The mappings can be generated using either `selective-alignment `_ or `pseudoalignment `_ against the transcriptome (with the ``--rad`` or ``--sketch`` flags, respectively). **Note**, however, that alevin-fry does not currently support RAD files aligned against a decoy-aware index, so that indices used for RAD file generation should be prepared without decoy sequnece. For a chromium v2 set of read files, the command would look like the following: .. code:: bash $ salmon alevin -lISR --chromium -1 -2 -o -i -p --sketch Given the output directory generated above, the next step is to let alevin-fry generate the permit list. First, we grab the 10x Chromium version 2 permit list (if we had Chromium v3 chemistry, we would use that permit-list instead): .. code:: bash $ wget https://umd.box.com/shared/static/jbs2wszgbj7k4ic2hass9ts6nhqkwq1p -O 10x_v2_permit.txt Now, we can use this permit list to scan the cell barcodes actually encountered in our reads and determine a set of cells that were likely present in our sample: .. code:: bash $ alevin-fry generate-permit-list --input --expected-ori fw --output-dir --unfiltered-pl 10x_v2_permit.txt Next, given the permit list and barcode mapping (which resides in the `` directory), we collate the original RAD file using the command below. .. code:: bash $ alevin-fry collate -i -r -t Finally, we quantify the collated rad file using the `cr-like` resolution strategy using the `quant` command below. .. code:: bash $ alevin-fry quant -i -m -t -r cr-like -o Note that with the exception of the `generate-permit-list` command, the other `alevin-fry` commands are designed to scale well with the number of provided threads. Thus, if you have multiple threads to use, then you can provide the appropriate argument to the `-t` option. Detailed information on the alevin-fry commands ----------------------------------------------- There are a (growing) number of different sub-commands for ``alevin-fry``. To learn more about the different commands an their options check the :ref:`commands` section of the documentation.