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-alignemnt or pseudoalignemnt against the transcriptiome (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:

$ salmon alevin -lISR --chromium -1 <read1_files> -2 <read2_files> -o <alevin_odir> -i <index> -p <num_threads> --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):

$ 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:

$ alevin-fry generate-permit-list --input <alevin_odir> --expected-ori fw --output-dir <fry_odir> --unfiltered-pl 10x_v2_permit.txt

Next, given the permit list and barcode mapping (which resides in the <fry_odir> directory), we collate the original RAD file using the command below.

$ alevin-fry collate -i <fry_odir> -r <alevin_odir> -t <num_threads>

Finally, we quantify the collated rad file using the cr-like resolution strategy using the quant command below.

$ alevin-fry quant -i <fry_odir> -m <tg_map> -t <num_threads> -r cr-like -o <fry_odir>

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 commands section of the documentation.