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  • assemble RNAseq data with Oases but so many transcripts in locus_1

    Hello everyone

    I'm doing transcriptome assembly work with Oases.
    The illumina data's read length is 100nt and its quality is pretty good. I filterred bed reads, trimmed bases of low quality and trimmed adapters. Then did the Oases_pipeline in this command:
    Code:
    nohup oases_pipeline.py -m 17 -M 71 -s 2 -g 27 -o Hg0912 -d " -short Hg-trim.fasta " &
    The final result in transcripts.fa seems not so good. The first locus "locus_1" has so many transcripts, which exceeds 600,000.
    >Locus_1_Transcript_1/656445_Confidence_0.000_Length_257
    TTATTTTCTTCCTGTTGTTTTCAGTACGAGCCAGTTGAGATGCGCGTGAGTTTATAAACA
    AAACCTGTGTCCCCGATTGGCCAGTAAGTAGCCGGCAACCGACACGGACGTTGTACTTGT
    ATTGAGCAAAGTTTATTCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACA
    TCCCGACGAATTGAGAACCTCTCCTTTTTGGGATAAAAAAAAAAAAAAAAAAAAAGTTGT
    ATTTTGTGTTTCAAAGT
    >Locus_1_Transcript_2/656445_Confidence_0.000_Length_505
    CATTCCTTGTATTCAAAAAAAAAAAAAAAAAAAAAAAAATGAAACACGTCAACAAAAAAA
    AAAAAAGGAACCCTTATTCTTAGAGAATTAAGACTTTTGCAAAAAAAAAAAAAAAAAAAA
    AAAAAAAAAAAAACATCCCGACGAATTGAGAACCTCTCCTTTTTGGGATAAAAAAAAAAA
    AAAAAAAAAAGCATGAAAAAAAAAAAATGCAGAAATCCCACTTTACTTTTAGATAAATAT
    TGCAAATTTGCGATACATAACATACATTAATTACATATAGGTAACTGTTTATTTTAAGGC
    AAATTCTTAGAAAAAACTAAGAAGTCCTGGATCAACTAAAAAATACAGCTCTCGAACGTC
    GCTCTTACAATTTTAAAACCAAGTTCCTTGAGTTAAAAATTGGAAAAAGTCGCGCTCGCT
    CCGCTCGCGATTTAGAAGCGATGTGCTTGTTTTTGCATTCGCCGGCCAACCAACAAAAAA
    TTATGGACGTTTGAGCTACACTTAT
    >Locus_1_Transcript_3/656445_Confidence_0.000_Length_407
    TCAAAAAAAAAAAAAAAAAAAAAAAAATGAAACACGTCAACAAAAAAAAAAAAAGGAACC
    CTTATTCTTAGAGAATTAAGACTTTTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAC
    ATCCCGACGAATTGAGAACCTCTCCTTTTTGGGATAAAAAAAAAAAAAAAAAAAAAGCAC
    GTCAACAAAAAAAAAAAAAGGAACCACATTGAATTTTGTGTTACGTCACTACTTTTAGGC
    ......
    And I pick out a long sequence to do blastx ,nothing similar found. But when blastn, the sequence can match to my species' genome sequences(my species do not have a complete genome sequneced, the matched subjects are something like BAC library).

    Is this normal?

  • #2
    Hi Minoru,

    I'm having the same problem. Did you find an answer for it? Thanks.

    Comment


    • #3
      Hi,
      Although our team has not seen this many transcripts under a given locus... what we have tended to do is set those loci with a given blastx hit as tier one transcripts (ie given them a higher ranking/priority over the other transcripts).

      On another note, not necessarily related to a scenario of the 6,000 transcripts under a single locus, but focussed on the results coming out of a transcriptome assembly: We found that the molecular biology protocols and depletions have a large impact on the resulting transcriptome assembly. In the early days before we had the protocols nailed down we found that a very large percent of our loci did not have blastx hits, as our molecular biology (ie not our bioinformatics) became more refined, we saw a much higher percent of loci matching to genes.

      I hope this is helpful.

      Jarret Glasscock
      Cofactor Genomics

      Comment


      • #4
        Thank you very much for the answer, it is very helpful.

        Mari

        Comment

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