Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • MiSeq amplicon sequencing Primers?

    Hi guys,

    I sent some soil samples to a commercial sequencing center. I do fungal ITS sequencing (300bpX 2 paired-end) Miseq.

    As you know, I use forward and reversed primers when I did PCR. I suppose Paired-end means sequencing twice, right? I suppose they should use forward primers to sequence once and reverse primer to sequence from another time.

    However, I received two mapping files from sequencing center. Both mapping file show they only used forward primer? I am wondering if this is normal?

    I know in our forward primer always better and accurate. Back to old days (Sanger sequencing) we only use forward primers to run sequencing reactions.

    If you guys are familiar with paired-end amplicon sequencing, I am wondering if it is normal to use only one primer to sequence both (paired).

    Thanks,

  • #2
    I assume you have done PCR wit ITS region specific primers with no Illumina overhang or full adapter sequences. In this case they must have prepared library by adapter ligation which will result in non-direction library so R1 and R2 can start for some fragments from forward direction and for others from reverse direction.

    You should check some reads from R1 and R2 sequence folders and you should see in both sequences starting with forward and reverse primers, unless they have been sequenced with custom primers or have been trimmed to remove PCR primers.

    Comment


    • #3
      Originally posted by nucacidhunter View Post
      I assume you have done PCR wit ITS region specific primers with no Illumina overhang or full adapter sequences. In this case they must have prepared library by adapter ligation which will result in non-direction library so R1 and R2 can start for some fragments from forward direction and for others from reverse direction.

      You should check some reads from R1 and R2 sequence folders and you should see in both sequences starting with forward and reverse primers, unless they have been sequenced with custom primers or have been trimmed to remove PCR primers.
      I don't quite understand, but I didn't do PCR. I gave them gDNA. They did the PCR and sequencing for me.

      They didn't give me raw fastq files such as R1 and R2. They only give me a combined fasta file. I only find adapter sequence and forward primer sequences in the fast file. I don't they if they removed reversed primers or they only did sequencing using forward primer.

      Comment


      • #4
        Paired end sequencing means that library fragments (in this case ITS amplicons) are sequenced from both ends so R1 and R2 of the fragment are in opposite direction. If sequencing cycles are longer than library fragment then R1 nad R2 can be merged (if there is enough overlap). You can ask the sequencing centre to provide at least the workflow and that should answer your question.

        Comment


        • #5
          Originally posted by nucacidhunter View Post
          Paired end sequencing means that library fragments (in this case ITS amplicons) are sequenced from both ends so R1 and R2 of the fragment are in opposite direction. If sequencing cycles are longer than library fragment then R1 nad R2 can be merged (if there is enough overlap). You can ask the sequencing centre to provide at least the workflow and that should answer your question.
          “R1 and R2 of the fragment are in oppossite direction". -- I know this. However, my question is when they sequence R1 and R2, do they have to use two primers? For example, R1 uses forward primer and R2 uses reverse primer.

          Or they even don't have to use the PCR primer? They use universal primers?

          Comment


          • #6
            R1 and R2 are sequenced with different primers. Sequencing primers could be one of Illumina sequencing primers or custom primers based on amplicon sequence. The primer choice is based on library prep workflow and design.

            Comment

            Latest Articles

            Collapse

            • 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
            • seqadmin
              The Impact of AI in Genomic Medicine
              by seqadmin



              Artificial intelligence (AI) has evolved from a futuristic vision to a mainstream technology, highlighted by the introduction of tools like OpenAI's ChatGPT and Google's Gemini. In recent years, AI has become increasingly integrated into the field of genomics. This integration has enabled new scientific discoveries while simultaneously raising important ethical questions1. Interviews with two researchers at the center of this intersection provide insightful perspectives into...
              02-26-2024, 02:07 PM

            ad_right_rmr

            Collapse

            News

            Collapse

            Topics Statistics Last Post
            Started by seqadmin, 03-14-2024, 06:13 AM
            0 responses
            34 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-08-2024, 08:03 AM
            0 responses
            72 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-07-2024, 08:13 AM
            0 responses
            81 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-06-2024, 09:51 AM
            0 responses
            68 views
            0 likes
            Last Post seqadmin  
            Working...
            X