Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Cutadapt - help.

    Hi,
    I am using cutadapt for removing the adapter sequence. I have 2 adapter sequence.

    RNA 5Adapter (RA5)
    5 GUUCAGAGUUCUACAGUCCGACGAUC
    RNA 3?Adapter (RA3)
    5 TGGAATTCTCGGGTGCCAAGG

    The 1st one is 5' adapter and 2nd is 3' adapter.

    I am using the following command line to remove the adapter seq.

    cutadapt -a TGGAATTCTCGGGTGCCAAGG -g GUUCAGAGUUCUACAGUCCGACGAUC input.fastq > output.fastq

    Length Distribution I get
    Mean sequence length: 32.49 ± 10.53 bp
    Minimum length: 16 bp
    Maximum length: 51 bp
    Length range: 36 bp
    Mode length: 51 bp with 2,852,626 sequences


    And I found that the 5' adapter has U instead of T. Will that be fine?

    I tried replacing U with T GUUCAGAGUUCUACAGUCCGACGAUC > GTTCAGAGTTCTACAGTCCGACGATC and tried removing adapter sequence.

    cutadapt -a TGGAATTCTCGGGTGCCAAGG -g GTTCAGAGTTCTACAGTCCGACGATC input.fastq > output.fastq

    Length Distribution I get
    Mean sequence length: 31.26 ± 11.29 bp
    Minimum length: 1 bp
    Maximum length: 51 bp
    Length range: 51 bp
    Mode length: 51 bp with 2,805,271 sequences

    I get varied length distribution in both the cases. Which one should I choose..
    First is the command that I am using is right??
    I am working on small RNA seq data.
    Kindly let me know.

    Thanks in advance.

    Regards
    Vishwesh

  • #2
    Reads don't come out of sequencers with 'U' in them, just 'T', so don't use 'U' for postprocessing reads. Some tools may understand it but most won't.

    And a varied length distribution is expected, is it not?

    Comment


    • #3
      Thanks for the response.
      Hope the command that I use is correct.
      Can you pls let me know how remove 5' adapter and 3' adapter using cutadapt??

      Regards
      Vishwesh

      Comment


      • #4
        Well, no, I don't use cutadapt. I can advise you on bbduk, though.

        bbduk.sh -Xmx1g in=input.fq out=trimmed3.fq ktrim=r literal=TGGAATTCTCGGGTGCCAAGG k=19 mink=12

        This will trim to the right when finding an adapter.

        bbduk.sh -Xmx1g in=trimmed3.fq out=trimmed3_5.fq ktrim=l literal=GTTCAGAGTTCTACAGTCCGACGATC k=19 mink=12

        This will trim to the left when finding an adapter.

        If you want to allow a mismatch, you can set "hdist=1".

        -Brian

        Comment


        • #5
          try skewer

          skewer is also qualified for trimming adapters from small-RNA reads.

          for your case, you may use the following command:

          Code:
          skewer -x TGGAATTCTCGGGTGCCAAGG small-RNA.fastq -1 | skewer -x GUUCAGAGUUCUACAGUCCGACGAUC -m head -r 0 -l 21 -L 25 -o trimmed -

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Strategies for Sequencing Challenging Samples
            by seqadmin


            Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
            03-22-2024, 06:39 AM
          • seqadmin
            Techniques and Challenges in Conservation Genomics
            by seqadmin



            The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

            Avian Conservation
            Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
            03-08-2024, 10:41 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, Yesterday, 06:37 PM
          0 responses
          11 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          10 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          51 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          68 views
          0 likes
          Last Post seqadmin  
          Working...
          X