Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Nextseq 500 amplicon length considerations?

    Hi guys,
    I'm planning a bacterial 16s rRNA amplicon sequencing project using Nextseq 500 with 300-cycles reagents. Now I'm trying to figure out how to choose an amplicon size and what the best run type is (single or paired end? If the latter, should the two paired end reads partially overlap?). Are there any basic "rules" or reliable primer sets available? Any suggestions/references would be appreciated.

  • #2
    It's best to run paired-end 2x150 with overlapping reads (design for at least 30bp overlap). This will greatly limit your set of available regions; if at all possible, I'd suggest 2x250 or 2x300 on a MiSeq.

    Comment


    • #3
      Originally posted by Brian Bushnell View Post
      It's best to run paired-end 2x150 with overlapping reads (design for at least 30bp overlap). This will greatly limit your set of available regions; if at all possible, I'd suggest 2x250 or 2x300 on a MiSeq.
      Thank you Brian. I will start from there

      Comment


      • #4
        Originally posted by psax View Post
        Hi guys,
        I'm planning a bacterial 16s rRNA amplicon sequencing project using Nextseq 500 with 300-cycles reagents. Now I'm trying to figure out how to choose an amplicon size and what the best run type is (single or paired end? If the latter, should the two paired end reads partially overlap?). Are there any basic "rules" or reliable primer sets available? Any suggestions/references would be appreciated.
        Commerically available V4 kits play well with 2x150. You could do a V1-V2 amplicon but you'll need longer reads to do that.

        As for the NextSeq, you'll have a really bad time unless you can base stagger your amplicons when you multiplex. Even with a healthy dose of PhiX you'll get poor quality as compared to the MiSeq. I suggest you read up on the primers used and homebrew your own kit.

        Comment


        • #5
          Furthermore, NextSeq has way more capacity than is needed for a 16s study, and currently has major problems with multiplexing; and the 4 "lanes" are not separated. So even if it supported longer reads, I would not recommend it for this purpose.

          Comment


          • #6
            Originally posted by bilyl View Post
            Commerically available V4 kits play well with 2x150. You could do a V1-V2 amplicon but you'll need longer reads to do that.

            As for the NextSeq, you'll have a really bad time unless you can base stagger your amplicons when you multiplex. Even with a healthy dose of PhiX you'll get poor quality as compared to the MiSeq. I suggest you read up on the primers used and homebrew your own kit.
            Thank you for your advices. With base stagger, do I still have to add 30-50% PhiX?

            Comment


            • #7
              I think you'll find more than a few posts recommending that you NOT use NextSeq for amplicon sequencing, even with PhiX. It simply doesn't work as well as MiSeq.

              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
              33 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