Help running snow crab genome through NCBI annotation pipeline

[laurahspencer]

Issue Category

Please select the category that best describes your issue:

• Server/Infrastructure (Raven, Klone, Gannet connectivity, performance, or storage issues)
• Bioinformatics Tools (Tool installation, container setup, pipeline errors, workflow issues)
• R/RStudio (Package installation, server connectivity, environment configuration)
• Data Management (File transfers, storage, backup procedures, data organization)
• Access Management (Account requests, permissions, authentication setup)
• Lab Meeting Topic (Suggestions for lab meeting presentations or discussions)
• Equipment/Hardware (Lab equipment issues, hardware problems)
• Other (Please describe in the issue description)

Computer System Information

Please provide details about the system you're working on:

Primary system (check all that apply):

• Raven (lab server)
• Klone (UW HPC cluster)
• Gannet (lab server)
• Personal computer (Mac/Windows/Linux)
• Other lab computer: ________________

Operating System (if known):

• Ubuntu/Linux
• macOS
• Windows
• Other: ________________

Connection method (if applicable):

• SSH/Terminal
• RStudio Server
• VPN
• Direct connection
• Other: ________________

Issue Description

Please provide a clear description of your issue or request:

NOAA has produced a ton of snow crab / tanner crab RNASeq data that I'd like to use to improve the existing snow crab genome assembly (GCA_016584305.1). Steven mentioned the NCBI annotation pipeline is public now. I'd love some help running the pipeline on the snow crab genome, if anyone is willing!

RNASeq data is from juveniles (homogenized whole animal), and adults (separate tissues - gill, hepatopancreas, muscle), and is currently stored on a NOAA server, but I'll happily rsync RNASeq data to where it needs to go.

Error Messages

If you encountered any error messages, please paste them here (use code blocks with triple backticks ```):

[Paste error messages here]

What You've Tried

Please describe any troubleshooting steps or solutions you've already attempted:

Expected Outcome

What were you trying to accomplish or what result were you expecting?

More complete annotation for genome assembly GCA_016584305.1.

Additional Context

Please provide any additional information that might be helpful:

• Relevant files or links:
• Timeline/urgency:
• Related issues:
• Screenshots (if applicable):

---

For Lab Members:

• If this is urgent, also post in the Roberts Lab Slack
• Check the Roberts Lab Handbook for existing documentation
• Browse existing GitHub Issues to see if similar issues have been resolved

[kubu4]

I'd love some help running the pipeline on the snow crab genome, if anyone is willing!

Is there anything specifically you need help with?

Are you stuck on part of the process?

FYI - We haven't used the pipeline, so may not be able to offer much insight, but we're definitely willing to help with whatever; just let us know.

[sr320]

Sam - do you have any experience with a different pipeline?

…

On Wed, Feb 18, 2026 at 3:42 PM kubu4 ***@***.***> wrote: *kubu4* left a comment (RobertsLab/resources#2399) <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2399*issuecomment-3923108247__;Iw!!K-Hz7m0Vt54!h4Jj5iWWQMtiNB-j-roLcSL6-tPdWbFpA_XDwKONbOgIxtuu1Zkjsu1QODYae5CeQAVdelqY8KZTgvl2YRvC85Y$> I'd love some help running the pipeline on the snow crab genome, if anyone is willing! Is there anything specifically you need help with? Are you stuck on part of the process? FYI - We haven't used the pipeline, so may not be able to offer much insight, but we're definitely willing to help with whatever; just let us know. — Reply to this email directly, view it on GitHub <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2399*issuecomment-3923108247__;Iw!!K-Hz7m0Vt54!h4Jj5iWWQMtiNB-j-roLcSL6-tPdWbFpA_XDwKONbOgIxtuu1Zkjsu1QODYae5CeQAVdelqY8KZTgvl2YRvC85Y$>, or unsubscribe <https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/ABB4PN5QD62FACC4FHPCXUT4MTFCJAVCNFSM6AAAAACVO574W2VHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZTSMRTGEYDQMRUG4__;!!K-Hz7m0Vt54!h4Jj5iWWQMtiNB-j-roLcSL6-tPdWbFpA_XDwKONbOgIxtuu1Zkjsu1QODYae5CeQAVdelqY8KZTgvl2bh0di3A$> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[kubu4]

The PASA pipeline I've been running for the coral project could definitely be used with the additional RNA-seq data and genome to update the genome.

However, I'm not certain how this would be incorporated with NCBI's genome annotation (which is how I interpreted this Issue). It might be feasible to get the new RNA-seq data submitted to NCBI SRA, then contact NCBI to have them update the genome with the new SRA data?

Also, I'm currently not seeing an available NCBI pipeline for eukaryotic genomes; only an available pipeline for prokaryotic genomes (PGAP).

[laurahspencer]

This is what I'm seeing as available for eukaryotes - https://github.com/ncbi/egapx

I'm not stuck, haven't started yet! I'm more looking for someone to team up with to help move the annotation forward. Interested? I've started moving RNASeq data over to Klone, and had planned on running it there with/without a teammate.

[sr320]

Could you run the PASA pipeline on hyak? I will dig around more to find NCBI code - they announced at PAG

…

On Wed, Feb 18, 2026 at 3:51 PM kubu4 ***@***.***> wrote: *kubu4* left a comment (RobertsLab/resources#2399) <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2399*issuecomment-3923153057__;Iw!!K-Hz7m0Vt54!j_3lPJXeIkQGPyq58eU8nZRAirtToQvl0OU3gG0hZSrY0Y0QjtXDl5tAI8upnmn06XEIuy3Ln_2knCGrIjkHzZI$> The PASA pipeline I've been running for the coral project could definitely be used with the additional RNA-seq data and genome to update the genome. However, I'm not certain how this would be incorporated with NCBI's genome annotation (which is how I interpreted this Issue). It might be feasible to get the new RNA-seq data submitted to NCBI SRA, then contact NCBI to have them update the genome with the new SRA data? Also, I'm currently not seeing an available NCBI pipeline for eukaryotic genomes; only an available pipeline for prokaryotic genomes (PGAP). — Reply to this email directly, view it on GitHub <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2399*issuecomment-3923153057__;Iw!!K-Hz7m0Vt54!j_3lPJXeIkQGPyq58eU8nZRAirtToQvl0OU3gG0hZSrY0Y0QjtXDl5tAI8upnmn06XEIuy3Ln_2knCGrIjkHzZI$>, or unsubscribe <https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/ABB4PNZKTWMAFOA2AWNCNXD4MTGGZAVCNFSM6AAAAACVO574W2VHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZTSMRTGE2TGMBVG4__;!!K-Hz7m0Vt54!j_3lPJXeIkQGPyq58eU8nZRAirtToQvl0OU3gG0hZSrY0Y0QjtXDl5tAI8upnmn06XEIuy3Ln_2knCGrTfdv3L8$> . You are receiving this because you commented.Message ID: ***@***.***>

[kubu4]

Can't run PASA on Hyak, unfortunatley. The process (specifically Trinity assemblies) generates too many intermediate files and causes it to exceed file count quotas.

Can run on Raven (which is what I've been doing), though.

[kubu4]

Also, what's the actual goal here? I mean, I understand you want to utilize that RNA-seq data, but to what end? Do you have a reason to believe that the existing NCBI genome annotations are insufficient?

I don't think you'll be able to update the existing NCBI genome annotations on the existing NCBI genome, as you're not the original submitter of the genome assembly.

[laurahspencer]

The goal is to improve annotations for the snow crab genome using the large amount of RNASeq data we've generated for the species, so that I can retain more data during differential expression analysis, and when identifying genetic variants in gene regions. Alignment to the genome is pretty darn good (~80%), but about half of the RNASeq align outside of known features (based on existing annotations), which suggests that annotations aren't complete.

[kubu4]

I don't think you'll be able to update the existing NCBI genome annotations on the existing NCBI genome, as you're not the original submitter of the genome assembly.

Never mind! Seems like you can update annotations:

https://github.com/ncbi/egapx?tab=readme-ov-file#submitting-annotation-for-an-existing-genbank-assembly

However, with that being said, you're probably going to want (need??) to have the RNA-seq available in the NCBI SRA...

So, maybe get the data submitted, then go through the pipeline? This would also make the process more reproducible, as you'd be using SRA accessions for the pipeline, instead of the RNA-seq FastQs, which other people wouldn't have access to.

[laurahspencer]

That sounds like a good idea, although I need to check to make sure I can post our new data on SRA- I'm just now working it up, it's not published yet.

Sam would you be interested in helping with this annotation project?

[kubu4]

Sam would you be interested in helping with this annotation project?

This is really a question for @sr320. 😃

[laurahspencer]

Actually, what I would really love help with is the software installation. Is it best to update the srlab container to include the NCBI annotation repo / software (where needed)? I haven't worked on Klone in a while

[kubu4]

Actually, what I would really love help with is the software installation

Yeah, I'll work on this.

[kubu4]

Alrighty, I have a Singularity (Apptainer) container and execution script which are functional - ran test annotation successfully.

Unfortunately, the srlab is out of storage on Hyak, so I can't transfer them.

Once that's resolved, I'll get things moved over and explain how to use them.

[kubu4]

Container and script have been added to Klone:

• /mmfs1/gscratch/srlab/containers/run_egapx.sh: Script to execute srlab-NCBI-EGAPx.sif container. This will create a cache directory, set a cache variable, and bind directories to the container.

  Example usage:

  ./run_egapx.sh \
  /opt/egapx/examples/input_D_farinae_small.yaml \
  -e singularity \
  -w ./work \
  -o ./output

  This requires that the srlab-NCBI-EGAPx.sif container is in the same directory as this script. Both can be copied to other directories, if desired.

• /mmfs1/gscratch/srlab/containers/srlab-NCBI-EGAPx.sif: Apptainer (Singularity) container. Can be executed by itself, but is better to use with the run_egapx.sh to ensure cache directory is set.

  To see additional help examples for using the srlab-NCBI-EGAPx.sif container:

  singularity run-help srlab-NCBI-EGAPx.sif

Finally, for info on how to run EGAPx: https://github.com/ncbi/egapx

EDITED: Added note about directory bindings.