Question About third_party_software Directory and Software Versions for CAMPAREE

[QianhuiWan]

Hello, I'm having trouble locating the third_party_software directory. Could I generate it myself?

Also, are there specific versions required for the following software used in CAMPAREE: STAR, Kallisto, Bowtie2, and Beagle?

 self.set_third_party_software()
  File "/home/miniconda3/envs/beers2_env/lib/python3.11/site-packages/camparee/expression_pipeline.py", line 309, in set_third_party_software
    beagle_filename = [filename for filename in os.listdir(ExpressionPipeline.THIRD_PARTY_SOFTWARE_DIR_PATH)
                                                ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
FileNotFoundError: [Errno 2] No such file or directory: '/home/miniconda3/envs/beers2_env/lib/python3.11/site-packages/third_party_software'

Thanks and best regards,
Qianhui

[tgbrooks]

Hi Qianhui,

Thanks for reach out to us. It looks like you're using miniconda3 to install beers, which I'm not experienced with. Can you walk me through your installation steps so that I can try to reproduce the problem?

The third_party_software directory is supposed to be installed automatically and contain everything you need. You can find it on git here but if it's not present in the expected location, something went wrong in the installation and possibly other aspects are wrong too.

Best,
Tom

[tgbrooks]

Also, can you please let me know what command you ran when you encountered this error? Thanks.

[QianhuiWan]

Hi Tom, Thank you so much for your prompt reply. I’ve attached the commands I used to install BEERS2 below:

conda create --name beers2_env python=3.11
conda activate beers2_env
# Install BEERS2 and dependencies
pip install git+https://github.com/itmat/BEERS2

The command I ran when I encountered the error is as follows:

run_camparee.py -c ${OUT_DIR}/simulated_reads/comparee_simulationConfig.yaml -r pmm7_sim -m serial -s 52 -d

Best regards,
Qianhui

[tgbrooks]

Hi Qianhui,

Thanks for that information. It looks like the problem is actually around CAMPAREE. I've gone and updated how CAMPAREE and BEERS2 are installed so that now when you install BEERS2 you'll get a complete CAMPAREE installation. Previously, it would only partially install CAMPAREE for the parts that BEERS2 uses. Try doing the BEERS2 installation again with a fresh environment. After that, both BEERS2 and CAMPAREE should be available for your use.

Let me know if anything else comes up.

Best,
Tom

[QianhuiWan]

Hi Tom, Thank you so much for your reply—it was very helpful! I have a group meeting tomorrow and need to prepare for it today, so I’ll reinstall and test again after the meeting. Apologies for the delay.

I also have another question regarding simulating RNA-Seq reads from repeat elements: do I need to add repeat annotations to the gene annotation .txt file?

Best regards,
Qianhui

[tgbrooks]

Hi Qianhui,

If it's in the reference genome, annotated, and you have reference samples expressing samples, then CAMPAREE will generated data corresponding to them automatically. If the repeat regions aren't in the genome, and/or the transcripts are unannotated, I think there are two approaches:

1. Update the genome and annotation to reflect those transcribed repeat regions. Make sure any insertions to the genome are reflected in the locations of any other genes in genome. Run CAMPAREE using those genomes and annotations. If the transcripts you are interested in didn't have any expression, then you can modify CAMPAREE's output to increase the expression of those transcripts. Then use this as input to BEERS2

2. Manually generate the molecule files for input to BEERS2 that contain the transcripts you're interested in. The format for that is described here. Note that you will need to include poly-A tails on the transcripts for them to be sequenced if using the example config files. PolyA tails get marked with "S" in the CIGAR string components (e.g. "40S" if using a 40 bp long polyA tail).

If you need help, please feel free to reach out to us via email (thobr@sas.upenn.edu) and we can setup a time to discuss in person.

[QianhuiWan]

Hi Tom,

I’ve reinstalled the BEERS2 package, and all third-party software dependencies are now working. However, I’ve encountered another issue with my .yaml configuration files. I’ve sent you an email regarding my script, and I’d be happy to arrange a time to discuss it with you whenever you’re available.

Thank you so much for your help!

Best regards,
Qianhui

[tgbrooks]

Hi Qianhui,

Unfortunately I didn't receive the email. Do you mind trying again at my other address: thobr@pennmedicine.upenn.edu ? Thanks!

Best,
Tom

[QianhuiWan]

Hi Qianhui,

Unfortunately I didn't receive the email. Do you mind trying again at my other address: thobr@pennmedicine.upenn.edu ? Thanks!

Best, Tom

Hi Tom,

I’ve resent my email. Thank you so much!

Best regards,
Qianhui

[QianhuiWan]

Hi Tom, thank you for your help! Sorry, I have so many questions. I still have a quick question about the unidentified FASTQ and BAM files in the output. Are they derived from CAMPAREE molecules but not properly annotated to genes?

Best regards,
Qianhui

[tgbrooks]

Hi QianhuiWan,

No, those are files where the sample barcode had read errors and so they were not assigned to a sample during demultiplexing. However, since the data is simulated, we do know what sample they came from, and so they are separated out by sample in case that is useful (for benchmarking demultiplexing, for example). In most studies, one can ignore those files.

[QianhuiWan]

Oh, I see. That’s great—thank you so much!