ERROR Running Detonate: RSEM EVAL on RNA SEQ assembly

Question

[nikelle.petrillo]

Hello,

I am quite new to RNA seq. I have assembled my first de novo assembled transcript via Trinity. I would like to assess the quality of my assembled transcript using RSEM-EVAL. I have installed Detonate, but I am having trouble running RSEM-EVAL without an error popping up. I do not have a reference transcriptome and I am not sure if I should be omitting something...

Here is the code I am using:

[npetrill@trogdor rsem-eval]$ ./rsem-eval-calculate-score ~/home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta ~/home/richardsonlab/AMMA_transcripts/trinity_out_dir/rsem_eval_1 1086  --transcript-length-parameters rsem-eval/true_transcript_length_distribution/human.txt -p 16

Invalid number of arguments!

The average contain length is 1086. I am not sure what the parameter -p 16 is used for and if I should be using a number other than 16. (just going off of http://deweylab.biostat.wisc.edu/detonate/vignette.html) In addition, since I do not have a reference transcriptome, should I be omitting the transcript length distribution? Also, not sure if this makes a difference but I assembled my transcript using paired end reads.

I appreciate any help I can get! Thanks so much for taking the time to help.

-Nikelle

Answer 1

[vladimir]

You have the wrong number of arguments in the wrong order.

If your reads are unpaired your command should look like this:

rsem-eval-calculate-score --transcript-length-parameters ParameterFile YourReads.fastq YourTrinityAssembly.fasta OutputPrefix AverageReadLength

If your reads are paired, you are expected to provide individual files with the left and right reads and the last argument is the average fragment length, instead of the average read length.

-p is the number of threads.

For more infor run

./rsem-eval-calculate-score --help

[nikelle.petrillo]

Thank you,

Do you know how I would go about finding an average fragment length? Is this the same as average contig length, because I have that number.

Also, I am having trouble finding an online guide that expands upon what "number of threads" mean. Could you expand upon this?

Thank you very much!

[nikelle.petrillo]

In addition, I tried a new string of commands:

npetrill@trogdor rsem-eval]$ ./rsem-eval-calculate-score --paired-end R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq Trinity.fasta /home/richardsonlab/AMMA_transcripts/trinity_out_dir/rsem_eval_1.fa 100

rsem-synthesis-reference-transcripts /home/richardsonlab/AMMA_transcripts/trinity_out_dir/rsem_eval_1.fa.temp/rsem_eval_1.fa 0 0 0 Trinity.fasta

rsem-synthesis-reference-transcripts : No such file or directory!

Please check if you have compiled the associated codes by typing related "make" commands and/or made related executables ready to use.

I am not sure what to make of this error message.