Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Adapter trimming NEBNext Library / MiSeq

    I have a 51 single-end reads generated with MiSeq using NEBNext Multiplex Oligos for Illumina.

    The sample sheet looks like this:

    Code:
    IEMFileVersion,4
    Investigator Name,FB
    Experiment Name,WT10104
    Date,11/27/2013
    Workflow,GenerateFASTQ
    Application,FASTQ Only
    Assay,TruSeq Small RNA
    Description,
    Chemistry,Default
    
    [Reads]
    51
    
    [Settings]
    ReverseComplement,0
    
    [Data]
    Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,Sample_Project,Description
    HS130333-1,,,,RPI3,TTAGGC,,
    HS130333-2,,,,RPI4,TGACCA,,
    HS130333-3,,,,RPI5,ACAGTG,,
    The Primer index manual can be found here.

    Sor for HS130333-1 file, according to the manual above the primer/adapter with index is:
    5 ́-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3 ́

    The document indicated that the expected index primer sequence read is TTAGGC which is the reverse complement of GCCTAA.


    My question is if I use `trim_galore` or `cutadapt` to trim the data, what is the parameter -a I should use?

    Is it the whole sequence above? Or first 5 ́-CAAGCAGAAGACGGCATACGAGAT?
    Or GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3 ́? (and what is 's' means here)

    Or the reverse complement of the each of above?

  • #2
    Since your sequence ends in ... GATC-s-T, you need to use its reverse complement, thus starting with AGATC.. This means you should be able to run Trim Galore in its default mode without specifying any -a because it is using just that sequence anyway.

    Comment


    • #3
      Hi, Thanks.

      But the reverse complement of

      5 ́-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3 ́

      Is this:
      GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG

      I.e. it doesn't start with "AGATC".
      Or trim_galore default adapter AGATCGGAAGAGC is no substring of the reverse complement above.

      Did I miss anything?

      Truly need your advice.

      Comment


      • #4
        You need to add an A to the start of the reverse complemented sequence which is a result of the A-tailing process in the Illumina library preparation protocol. Then the start of both sequence will match up perfectly which is what your want to use. Hth

        Comment


        • #5
          Thanks a million!
          So in the trimming process, I don't have to care about the index sequence "GCCTAA" or it's reverse complement "TTAGGC". Am I right?

          Comment


          • #6
            That's right, the index is only relevant to sort out different barcodes; for adapter trimming purposes it is sufficient to specify only the start of the adapter which all indexed adapters have in common. Good luck!
            Last edited by fkrueger; 01-27-2014, 01:37 AM. Reason: sounds better

            Comment


            • #7
              Originally posted by fkrueger View Post
              Since your sequence ends in ... GATC-s-T, you need to use its reverse complement, thus starting with AGATC.. This means you should be able to run Trim Galore in its default mode without specifying any -a because it is using just that sequence anyway.


              You save my life. If you don't mind one last question.
              How can I know whether or not to use the reverse complement adapter for trimming?

              Any other alternative than GATC-s-T ending?

              Comment


              • #8
                I think that all Illumina adapters end in exactly this sequence, you just need to draw it out once and it will become very obvious (I am certain a sketch of this can be found in of the other threads here on SeqAnswers). Small RNA adapter are different, but for all TruSeq adapters etc you should be fine using the defaults.

                Comment


                • #9
                  Thank you.

                  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