Using Trinity with case/control data
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9.7 years ago
bdeonovic ▴ 210

I would like to do RNA-seq differential expression analysis on an experiment run on 8 pigs (4 cases and 4 controls, not matched). I've already done the tophat/cuffdiff approach using the pig genome. My PI also wants me to try out Trinity (without using the pig genome sequence).

I noticed that trinity can take in multiple fastq files at a time. How should I run my Trinity run(s)? Do I do 8 separate Trinity runs? Two runs for the two conditions?

Thanks

sequencing Trinity RNA-Seq • 2.5k views
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9.7 years ago
st.ph.n ★ 2.7k

If you're looking to do a DE analysis on your Trinity results, then provide all of your fastq's at the same time. Take note if you have PE reads or SE reads. They are supplied differently to Trinity on the command line. This will create a single de novo assembly. If you assemble each sample separately, you'll have trouble with the RSEM step when aligning the raw reads back to the assembly to get abundance estimates. You can do individual runs, if you simply just want a de novo assembly for that sample. But for the DE analysis, you'll need to create a single assembly, combining all samples as inputs.

Example:

if PE: cat all your read 1's, and cat all your read 2's

Trinity --seqType fq --JM 50G --left reads_1.fq  --right reads_2.fq --CPU 6

For the DE analysis downstream you'll be able to specify replicates, and sample names.

If all fastq's are SE there is a --single option to use instead of specifying read orientation.

Check out the Trinity site; and for full usage there is a command line parameter: --show_full_usage

Hope this helps.

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This is what I ended up doing, except instead of cat-ing the reads together I provided a list of them (separated by a space) since Trinity says I can provide 1 or more fastq files.

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You can provide a list of file names for the --left and --right command line parameters but that list of names should be a comma separated list, not space separated. I fear that in this case Trinity may have taken the first file in each list and ignored the rest.

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The Trinity documentation is very shoddy...This is what it says:

If paired reads:
#      --left  <string>    :left reads, one or more (separated by space)
#      --right <string>    :right reads, one or more (separated by space)
#
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I've always just concatenated the files in respect to read orientation.

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