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TRINITY:Input (Fastq) and Output files (GFF)

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  • TRINITY:Input (Fastq) and Output files (GFF)

    Dear All,

    I am a new user for Trinity. I have 3 fastq files (2 paied end + 1 single end), From the documentation, it is writen that It can accept either single ends (--single). or pair ends (--right and -- left options). can I run the 3 files in a single run, or shall I do 2 separate runs 1 for single and other for paired ? can I merge the output if it should be 2 separate run?

    Another question, How can I parse the output to GFF files?

    Thank you in advance,
    Best Regards,

  • #2
    You've probably already figured this out, but the Trinity documentation describes what you should do:

    http://trinityrnaseq.sourceforge.net/#running_trinity

    If you have both paired and unpaired data, and the data are NOT strand-specific, you can combine the unpaired data with the left reads of the paired fragments. Be sure that the unpaired reads have a /1 as a suffix to the accession value similarly to the left fragment reads. The right fragment reads should all have /2 as the accession suffix. Then, run Trinity using the —left and —right parameters as if all the data were paired.

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    • #3
      Question about Trinity.pl paired and unpaired read input

      Hi all,
      I have a similar question about read input to Trinity.pl (version 2012-10-05): I used simultaneously both fastq paired reads, with parameters "--left" and "--right", and fastq unpaired reads (with read names ending both in /1 and /2), with parameter "--single".

      The program did not complain and returned an assembly, that looks reasonably legit. Has the program used all reads in the input, both paired and unpaired, or just one set? or some other combination?

      Thanks.

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      • #4
        If you have manged to save the run-time output from Trinity then at the top it will say what files went into the 'left' and 'right' working files. Look for something like the following.
        Code:
        Wed Jan  9 13:15:19 EST 2013
        Converting input files. (both directions in parallel)CMD: /group/apps/bioinformatics/apps/trinityrnaseq_r2012-10
        -05/util/..//trinity-plugins/fastool/fastool --illumina-trinity --to-fasta /

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        • #5
          Thanks a lot. Yes, I still had the Trinity log and checked the entries and it appears only the paired fastq files were used, from parameters "--left" and "--right". The unpaired input in the "--single" parameter was not used.

          At the same location of the log, I also found the following entry:

          "Done converting input files.CMD: cat left.fa right.fa > single.fa"

          This doesn't mean that Trinity is not making use of pair information, does it?
          I hope not.

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          • #6
            Trinity should be using the '/1' and '/2' of the Illumina file names in order to use pairing information.

            Undoubtedly you've already read the Trinity FAQ but I'll repeat part of it here.

            How do I combine multiple libraries in a single Trinity run? Or, how do I combine paired and single reads?
            If you have RNA-Seq data from multiple libraries and you want to run them all through Trinity in a single pass, simply combine all your left.fq files into one left.fq file, and combine all right.fq files into one right.fq file. Then run Trinity using these separately concatenated left and right input files.

            If you have additional singletons, add them to the .fq file that they correspond to based on the sequencing method used (if they're equivalent to the left.fq entries, add them there, etc).

            There is no good way to combine strand-specific data with non-strand-specific data, unless you decide to treat the entire data set as non-strand-specific.
            As I recall from discussion on the Trinity users' mailing list if you do know know what strand your single sequences are from then you should just add them to 'left.fa' with the '/1' as part of their names.

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            • #7
              Great, thanks. Yes, I had seen a similar description in the Trinity documentation page on sourceforge.net, but was not sure what happened during the run :

              "If you have both paired and unpaired data, and the data are NOT strand-specific, you can combine the unpaired data with the left reads of the paired fragments. Be sure that the unpaired reads have a /1 as a suffix to the accession value similarly to the left fragment reads. The right fragment reads should all have /2 as the accession suffix. Then, run Trinity using the —left and —right parameters as if all the data were paired. "

              My unpaired reads are not strand-specific, so I'll add them all to the left set with suffix /1 (even though they derive from broken pairs, and they currently have both /1 and /2 suffices).

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