Improving PacBio barcode-variant mapping (subassembly) using multiple sequence alignment.
To use, git clone or download and unzip the PacRAT code.
git clone https://github.com/dunhamlab/PacRAT.git
From the PacRAT directory, run:
qsub ./driver_msa.sh
to run PacRAT with the example data provided in this repository. To run with different data, update the driver_msa.sh script with the location of the input and output files, described below.
Scripts can run locally, although we recommend using a cluster/job submission system to optimize memory usage.
To set up your environment, install Anaconda and ensure it is working on your computer. Once Anaconda is installed, you should be able to run the driver_msa.sh script. If you run the Python script on its own (without using the driver_msa.sh script, be sure to load the environment conda env create --file msaccs_env.yml and conda activate msa_ccs beforehand to ensure all packages are installed, compatible, and activated.
The msa_pacbio.py script utilizes the multiple sequence aligner, MUSCLE and Needle through
EMBOSS.
To install MUSCLE, extract the zipped MUSCLE file tar -zxvf muscle_filename_here.tar.gz. The MUSCLE software is ready to run as soon as it is unzipped. For the EMBOSS file, unzip it tar -zxvf emboss_filename_here.tar.gz. Through terminal, go to the unzipped EMBOSS directory and type ./configure. When that is finished, type make (this may take 5-10 minutes). The software needed through EMBOSS is called needle, which is located in EMBOSS-versionX/emboss/needle.
In your driver script (you can use driver_msa.sh to run your program; be sure to comment out Section 1 and uncomment Section 2), specify the location of each software using the -m and -n options. You will also need to specify the input files (--highQual and --inputSeqs), as well as your working directory (-d) and output file (-o).
Parameter Descriptions
| Option | Description |
|---|---|
| -d,--directory | Specify working directory where intermediate and output files will be located |
| -o,--out | Specify the name of the final output file (default = Seq_barcodes_aligned.txt) |
| --highQual | File of barcode-variant association, where the variant is the highest quality read |
| --inputSeqs | Raw barcode, variant, and quality of sequences |
| -c,--cutoff | Minimum number of CCS reads needed in order to retain reads associated with specific barcodes (default = 1) |
| -t,--threshold | Minimum frequency threshold for calling consensus reads (default = 0.6) |
| -s,--stats | Option to generate alignment stats. Currently outputs below_threshold_Ncount.txt, barcodes_below_cutoff.txt files, and ccs_count_per_barcode.txt; see below for details |
| -v,--verbose | Print verbose debug output |
| -m,--muscle | Location of compiled/extracted MUSCLE program |
| -n,--needle | Location of compiled Needle program |
| --cont | If program is disrupted or aborts, enabling this feature will allow user to continue with unprocessed reads. Previously processed reads will not be reprocessed |
| -r,--rmint | Removes intermediate alignment files |
Input files:
This script requires two input files. The input files for both --highQual and --inputSeqs file should be tab-delimited file, where the first column is the barcode and the second column is the associated read. You can generate both these files following the pipeline described in the AssemblyByPacBio repository. The --inputSeqs file will be generated from the extractBarcodeInsertPairs_moreQC.py script, and the --highQual file will be generated from the unifyAssignment.py script.
Output files:
- The output file, dictated by the file name you specify for
--out, is a tab-delimited file, where the first column is the barcode and the second column is the aligned associated read. - The script will also produce a file called
progress_file.txt. This file is generated when a barcode is finished being processed. If your script is killed in the middle of a run, you can include the--contoption the next time you run the driver script, and it will not reprocess barcodes that were already processed in the last run. - The
barcodes_below_cutoff.txtfile lists barcodes that were not processed because they did not meet the read cutoff specified by--cutoff, if the--statsoption is included. You can double check that the script is working properly by adding together the number of barcodes in your output file (from--out) and the number of barcodes in thebarcodes_below_cutoff.txtfile. If the--statsoption is not included, thebarcodes_below_cutoff.txtfile will not be generated. - The
below_threshold_Ncount.txtfile returns the number of barcodes that contained ambiguous sites if the--statsoption is included. Sites are determined to be ambiguous if nucleotides in the same position do not pass the majority threshold specified by the--thresholdparameter. If the--statsoption is not included, thebelow_threshold_Ncount.txtfile will not be generated. - The
ccs_count_per_barcode.txtfile returns the number of CCS reads associated with each barcode. File is created if the--statsoption is included. If the--statsoption is not included, theccs_count_per_barcode.txtfile will not be generated.
Example input/output files:
The example input files provided are located in the input folder and hold the --highQual and --inputSeqs files from a subset (1000 barcodes) of the simulated H2B library. These example files run in ~ 1 min on the GS cluster.