Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • 16S sequencing from total RNA?

    I'm working with a researcher that wants to identify the bacterial communities present in human dental pulp tissue, blood, and saliva from isolated RNA. What is the advantage (or disadvantage) from starting with RNA versus DNA? Typical 16S workflows use DNA as input with targeted 16S rRNA V3/V4 region amplification for library generation and long-read sequencing. This researcher has already performed DNA-Seq in a previous study but now "wants to confirm results through RNA"...

  • #2
    Well, on DNA level you get everything, dead or alive. On RNA level, you get the viable bacterial community, which can be very different in sites of active immune response for example.

    Comment


    • #3
      Thanks for your reply.

      Yes, RNA-seq can provide what's transcriptionally active at the time of sampling. I'm wondering if there is an RNA workflow similar to 16S metagenomic sequencing for taxonomic classification. Perhaps this is more of an analysis tool question than methodology, however, I'm proposing total RNA isolation, host depletion and bacterial enrichment prior to library generation since these samples are suspected to be of low bacterial load.

      Does anyone have experience or suggestions for bacterial taxonomic characterization using RNA-seq?

      Comment


      • #4
        No, the process is much simpler as you think. You simply isolate total RNA and reverse transcribe that into cDNA using random primers. Than you do your normal 16S amplicon workflow on that cDNA. You just use cDNA instead of genomic DNA as input. That way you get a 16S taxonomic dataset of the active microbiota.
        What you suggest to do is a metatranscriptome which is a whole different approach.
        Last edited by Genetic Librarian; 07-17-2018, 05:22 AM.

        Comment


        • #5
          Should I still use a host depletion and bacterial enrichment method prior to reverse transcription if I only want to target bacteria for sequencing? I’m concerned that the sequencing reads will largely be human if not. Thermo has a MICROBEnrich and MICROBExpress line for host separation and bacteria mRNA enrichment. Illumina has a 16S rRNA workflow on the MiSeq using V3/V4 primer pair for amplification that I would like to use for this project if compatible.

          Comment


          • #6
            If you use an amplicon approach (16S v3v4) you do not need any form of depletion, because you just amplify bacterial 16S sequences . The primers are specific, so that it does not matter how much of host RNA is present. The same reason why you do not get host reads in your 16S datasets from DNA.
            Host depletion and bacterial enrichment is something you need for metatranscriptomes, not for 16S.

            Comment


            • #7
              Of course - makes sense! I think I'm overthinking this one

              Thanks for your input!

              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