Salmon Quantification cannot find read files

[Dwight Causey]

Hi all,

I'm trying to determine my transcript abundance using trinity and salmon, using this code:

~/data/programs/trinity/util/align_and_estimate_abundance.pl --seqType fq  \

        --samples_file ~/scratch/trinity_output/design_quant.txt --transcripts ~/scratch/trinity_output/Trinity.fasta \

        --est_method salmon --trinity_mode --prep_reference --thread_count 10

I get the following error:

Error, cannot locate file: 5CHC_4659_left.fastq.gz.P.qtrim.gz as specified in samples file: ~/scratch/trinity_output/design_quant.txt at ~/data/programs/trinity/util/align_and_estimate_abundance.pl line 943, <$fh> line 1.

The read files I have are located in the same directory as the design_quant.txt file (~/scratch/trinity_output/), so I cannot figure out why it is looking for them in the trinity directory. Seems like this should be something straightforward that I am just overlooking.

Any assistance would be greatly appreciated!

Cheers,

Dwight

[Brian Haas]

Hi Dwight,

In your samples file, please specify the full paths to the locations of each of the input fastq files.   If the fastq files are in your current working directory, then just specifying the base filenames alone would be fine, otherwise, full paths are required.

If that's not the case, then there's a bug I need to investigate.

best,

~b

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[Dwight Causey]

Hey Brian,

That did the trick! Thank you for the quick reply!

Cheers,

Dwight

[Dwight Causey]

Hey Brian,

I didn't know if I should start a new thread, but as a hopefully quick follow-up, I am getting an error when running salmon that seems like it isn't properly producing the its .json file. I've run the following code:

/home/dcausey/projects/def-manzonri/dcausey/lwf_data/programs/trinity/util/align_and_estimate_abundance.pl --seqType fq  \

        --samples_file design_quant.txt --transcripts Trinity.fasta \

        --est_method salmon --trinity_mode --prep_reference --thread_count 10 --output_dir transcript_quant

Which produces the following error:

CMD: salmon quant -i /scratch/dcausey/trinity_output/Trinity.fasta.salmon.idx -l IU -1 /scratch/dcausey/trinity_output/5CHC_4659_left.fastq.gz.P.qtrim.gz -2 /scratch/dcausey/trinity_output/5CHC_4659_right.fastq.$

Version Info: This is the most recent version of salmon.

### salmon (selective-alignment-based) v1.3.0

### [ program ] => salmon

### [ command ] => quant

### [ index ] => { /scratch/dcausey/trinity_output/Trinity.fasta.salmon.idx }

### [ libType ] => { IU }

### [ mates1 ] => { /scratch/dcausey/trinity_output/5CHC_4659_left.fastq.gz.P.qtrim.gz }

### [ mates2 ] => { /scratch/dcausey/trinity_output/5CHC_4659_right.fastq.gz.P.qtrim.gz }

### [ output ] => { 5CHC_4659 }

### [ threads ] => { 10 }

### [ validateMappings ] => { }

Logs will be written to 5CHC_4659/logs

[2020-11-12 10:19:00.365] [jointLog] [info] setting maxHashResizeThreads to 10

[2020-11-12 10:19:00.366] [jointLog] [info] Fragment incompatibility prior below threshold.  Incompatible fragments will be ignored.

[2020-11-12 10:19:00.366] [jointLog] [info] Usage of --validateMappings implies use of minScoreFraction. Since not explicitly specified, it is being set to 0.65

[2020-11-12 10:19:00.366] [jointLog] [info] Usage of --validateMappings implies a default consensus slack of 0.2. Setting consensusSlack to 0.35.

[2020-11-12 10:19:00.366] [jointLog] [info] parsing read library format

[2020-11-12 10:19:00.366] [jointLog] [info] There is 1 library.

Exception : [Error: The index version file /scratch/dcausey/trinity_output/Trinity.fasta.salmon.idx/versionInfo.json doesn't seem to exist.  Please try re-building the salmon index.]

salmon quant was invoked improperly.

For usage information, try salmon quant --help

Exiting.

I tried running just the reference prep step, which "runs" and I don't get any reported errors, but I also don't see any output when the code finishes running either. So, it seems like something has gone wrong with the reference prep?

/home/dcausey/projects/def-manzonri/dcausey/lwf_data/programs/trinity/util/align_and_estimate_abundance.pl --transcripts Trinity.fasta --est_method salmon --trinity_mode --prep_reference

Cheers,

Dwight

[Brian Haas]

Hi Dwight,

I haven't worked with the latest salmon.  The one we use in Trinity is salmon v1.0.  Want to try that version and see if it works?

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[Dwight Causey]

Hi Brian,

I downgraded to salmon v1.0.0 and I'm still getting the same error with the json filing missing.  

CMD: salmon quant -i /scratch/dcausey/trinity_output/Trinity.fasta.salmon.idx -l IU -1 /scratch/dcausey/trinity_output/5CHC_4659 -p 10 -validateMappings

Version Info: ### PLEASE UPGRADE SALMON ###

### A newer version of salmon with important bug fixes and improvements is available. ####

###

The newest version, available at https://github.com/COMBINE-lab/salmon/releases

contains new features, improvements, and bug fixes; please upgrade at your

earliest convenience.

###

Sign up for the salmon mailing list to hear about new versions, features and updates at:

https://oceangenomics.com/subscribe

###

### salmon (mapping-based) v1.0.0

### [ program ] => salmon

### [ command ] => quant

### [ index ] => { /scratch/dcausey/trinity_output/Trinity.fasta.salmon.idx }

### [ libType ] => { IU }

### [ mates1 ] => { /scratch/dcausey/trinity_output/5CHC_4659_left.fastq.gz.P.qtrim.gz }

### [ mates2 ] => { /scratch/dcausey/trinity_output/5CHC_4659_right.fastq.gz.P.qtrim.gz }

### [ output ] => { 5CHC_4659 }

### [ threads ] => { 10 }

### [ validateMappings ] => { }

Logs will be written to 5CHC_4659/logs

[2020-11-13 04:19:43.362] [jointLog] [info] Fragment incompatibility prior below threshold.  Incompatible fragments will be ignored.

[2020-11-13 04:19:43.362] [jointLog] [info] Usage of --validateMappings implies use of minScoreFraction. Since not explicitly specified, it is being set to 0.65.

[2020-11-13 04:19:43.362] [jointLog] [info] Usage of --validateMappings implies a default consensus slack of 0.2. Setting consensusSlack to 0.35.

[2020-11-13 04:19:43.362] [jointLog] [info] parsing read library format

[2020-11-13 04:19:43.362] [jointLog] [info] There is 1 library.

Exception : [Error: The index version file /scratch/dcausey/trinity_output/Trinity.fasta.salmon.idx/versionInfo.json doesn't seem to exist. Please try re-building salmon index.]

salmon quant was invoked improperly.

For usage information, try salmon quant --help

Exiting.

I tried running just the reference prep step as indicated in my previous post with salmon v1.0.0 and it still "runs" for about a second with no error, but there does not appear to be any output from the code within the working directory.

/home/dcausey/projects/def-manzonri/dcausey/lwf_data/programs/trinity/util/align_and_estimate_abundance.pl --transcripts Trinity.fasta --est_method salmon --trinity_mode --prep_reference

[Brian Haas]

I wonder  if it's relying on earlier checkpoints.

Can you copy the Trinity.fasta file to a new directory and try running it there?

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[Dwight Causey]

Hi Brian,

I copied the Trinity.fasta and all the read files into a new directory and ran it from there, but still getting the same error. I'm not sure if it is properly prepping the reference for quantification? I have attached the output from the code I ran if you were so inclined to look through it.

Cheers,

Dwight

[Brian Haas]

Hi Dwight,

It looks like you're using the right version of salmon, but I'm not sure why it's giving errors like it is.  We've been using this version for a long time now and no one else has complained about this specific issue, so it's very curious.

We'll look into upgrading salmon for the next Trinity release, but that doesn't solve your current problem.  Note, we support kallisto, so you could try running that instead of salmon and see how that goes. 

best,

~b

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[Brian Haas]

Also, I should mention that we support docker and singularity (and I prefer singularity).  If you're able to run our singularity image, it comes with everything installed (that's been fully tested and working) and should ideally just work for you too.

best,

~b