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  • Appropriate Adapter Sequence fora trimming? RNAseq Transcriptome

    For previous studies I have contracted out both Illumina sequencing and RNAseq analysis of transcriptomes. An updated reference genome was added to NCBI this past year, and I want to take previously sequenced fastq files and realign them to the new reference genome in galaxy using Tophat and Cufflinks. I contacted the sequencing facilities to understand their protocol to determine if adapter sequences had been removed. They were unclear whether or not the trimming had already been executed, and their response time to my questions is lagging. They have however sent me this email.

    "We performed a polyA selection with a kit from Ambion: MicroPoly(A) Purist Kit; required 1000ng as starting material

    We used a cDNA kit from NuGEN: Ovation RNA-Seq System V2; we added 1ng of mRNA into the reaction. There is a SPIA adaptor sequence you will most likely want to trim off the 5' ends.
    We used an in house cocktail for the Illumina libraries (combination of kits from Lucigen and NEB); we initiated the Illumina libraries with 500ng of cDNA. We used the indexed Illumina adaptors:

    Multiplexing Adapters
    5' P-GATCGGAAGAGCACACGTCT
    5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT

    Multiplexing PCR Primer 1.0
    5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

    Multiplexing PCR Primer 2.0
    5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

    PCR Primer, Index 1
    5’ CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTC

    Let me know if you have more questions.

    Thanks!
    Catrina


    Index Sequence - XXXXXX
    Sample Library
    GCCGCG CPAM-RS15-RS15-V1 CPAM-RS15-RS15-V1-cDNA-1-lib1a
    GCTCCA CPAM-RS14-RS14-CS1 CPAM-RS14-RS14-CS1-cDNA-1-lib1a
    GGCACA CPAM-RS9-RS9-CS1 CPAM-RS9-RS9-CS1-cDNA-1-lib1a
    GGCCTG CPAM-RS12-RS12-V1 CPAM-RS12-RS12-V1-cDNA-1-lib1a
    GTCCGC CPAM-RS11-RS11-CS1 CPAM-RS11-RS11-CS1-cDNA-1-lib1a
    TAATCG CPAM-RS11-RS11-V1 CPAM-RS11-RS11-V1-cDNA-1-lib1a
    TACAGC CPAM-RS16-RS16-V1 CPAM-RS16-RS16-V1-cDNA-1-lib1a
    TATAAT CPAM-RS13-RS13-CS1 CPAM-RS13-RS13-CS1-cDNA-1-lib1a
    Index Sequence - XXXXXX Sample Library
    CACCGG CPAM-RS12-RS12-CS1 CPAM-RS12-RS12-CS1-cDNA-2-lib2a
    CCTTAG CPAM-RS9-RS9-V1 CPAM-RS9-RS9-V1-cDNA-2-lib2a
    CTCAGA CPAM-RS8-RS8-CS1 CPAM-RS8-RS8-CS1-cDNA-2-lib2a
    CTGCTG CPAM-RS8-RS8-V1 CPAM-RS8-RS8-V1-cDNA-2-lib2a
    GACGGA CPAM-RS13-RS13-V1 CPAM-RS13-RS13-V1-cDNA-2-lib1a
    GATATA CPAM-RS14-RS14-V1 CPAM-RS14-RS14-V1-cDNA-2-lib1a
    GATGCT CPAM-RS15-RS15-CS1 CPAM-RS15-RS15-CS1-cDNA-2-lib1a
    GTAGAG CPAM-RS16-RS16-CS1 CPAM-RS16-RS16-CS1-cDNA-2-lib1a "

    I want to make sure that there is no contamination of adapter sequences, even though the quality of my reads is high. I have tried creating a fasta file using the Multiplexing Adapters sequences listed above, and attempting trimming using Scythe, however the output file always is empty. Am I using the correct sequences? Do I need to trim individual fastq files using their appropriate indexes? I am still new to the process, so any advice would be appreciated.

  • #2
    I recommend creating a fasta file with all the sequences, except for the one with XXXXXX, make a copy of that with each index - GCCGCG, GCTCCA, etc. Then trim all of your reads with the same fasta file. Sometimes there is confusion about which orientation the adapters will be read in, so it may also be useful to try the reverse-complements as well if scythe doesn't do that automatically.

    If the reads have never been trimmed in any way, they will all be the same length.

    Comment


    • #3
      Brian,

      Thanks for the advice.

      I am still having problems with trimming using the tool scythe in a local host galaxy. I went ahead and created a fasta file containing all of the adapters and their reverse sequences. The resulting output has a description of "no peek" and is empty. The fasta file is in the proper format with each adapter identification line starting with a ">" and a following line of sequence containing only basepair letters (see attached).

      Is this an internal problem with scythe downloaded to galaxy? would another tool be more appropriate for my need of trimming out adapters? What causes this no peek result?
      Attached Files

      Comment


      • #4
        Your adapter file appears to be valid. Unfortunately, I don't use Galaxy or Scythe, so I'm not sure what the problem might be, or what "no peek" is supposed to mean. If you are able to process your data on the command line, I suggest you try running BBDuk, like this:

        bbduk.sh in=reads.fastq out=trimmed.fastq ref=adapters.txt ktrim=r k=23 mink=11 hdist=1

        Comment


        • #5
          I don't have access to a galaxy with scythe installed but I think this may be a problem with your local galaxy. Have you asked your local galaxy admins/support for help?

          There are many tools that can trim but you will need to work outside galaxy.

          Comment

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