Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • stranded bacterial RNA-seq

    We did a stranded RNA-seq library prep on a bacterial sample (Mycobacterium tuberculosis). When using this kit for human/mouse/fly, the correct strand is always "second" or "reverse" strand (different tools have different nomenclature). This time, I am getting 90% of the reads on the other strand. It doesn't make much sense to me that the reads would somehow be on the wrong strand. At worst, they should be 50/50. Do the bacterial annotations have different assumptions? Should I be concerned?
    Last edited by id0; 04-07-2015, 01:26 PM.

  • #2
    Hi,
    a lot of strand-specific RNA-Seq kits produce "reverse" reads. See http://seqanswers.com/forums/showthread.php?t=50459 and http://seqanswers.com/forums/showthread.php?t=50711.
    I'd guess it is more a library-prep issue than an annotation issue; so I don't think you should be concerned.

    Comment


    • #3
      Originally posted by Michael.Ante View Post
      Hi,
      a lot of strand-specific RNA-Seq kits produce "reverse" reads. See http://seqanswers.com/forums/showthread.php?t=50459 and http://seqanswers.com/forums/showthread.php?t=50711.
      I'd guess it is more a library-prep issue than an annotation issue; so I don't think you should be concerned.
      I think you misunderstood my question. I am familiar with the kit and I've used it for other species many times. The reads are always "reverse". Now I did bacteria and they are not "reverse".

      Comment


      • #4
        Reverse while mapping, or reverse when doing an assembly?

        Comment


        • #5
          What kit are you using?

          Yes, if you use the same kit in the same way your strandedness should remain the same.

          My first suspicion would be that the annotation for your Mycobacterium is wrong.

          --
          Phillip

          Comment


          • #6
            Originally posted by pmiguel View Post
            What kit are you using?

            Yes, if you use the same kit in the same way your strandedness should remain the same.

            My first suspicion would be that the annotation for your Mycobacterium is wrong.
            The kit is ScriptSeq Complete Gold Kit (http://www.illumina.com/products/scr...demiology.html). It even says on the website: "choose the FR=secondstrand option". That always works fine for me.

            I am also concerned that the annotation is wrong, but I am using the references from Ensembl (http://bacteria.ensembl.org/mycobact..._2/Info/Index/). I assume their info would be consistent across all species. I never ran into any issues like this with non-bacterial references.

            Comment


            • #7
              It would be odd to have entire genome annotation wrong from Ensembl (but strange things do happen). Are you using the GFF file Ensembl is providing?

              Can you compare the annotation from NCBI: http://www.ncbi.nlm.nih.gov/genome/?...culosis+H37Rv?

              Comment


              • #8
                When you write "This time, I am getting 90% of the reads on the other strand." what do you mean by "getting". I presume you use some program or browser to derive this figure? What program or browser did you use? Was it the same one you used for previous projects?
                --
                Phillip

                Comment


                • #9
                  Originally posted by GenoMax View Post
                  It would be odd to have entire genome annotation wrong from Ensembl (but strange things do happen). Are you using the GFF file Ensembl is providing?

                  Can you compare the annotation from NCBI: http://www.ncbi.nlm.nih.gov/genome/?...culosis+H37Rv?
                  I used the Ensembl GFF. I also tried the NCBI GFF. Similar result with both.

                  I use Picard CollectRnaSeqMetrics to collect the stats. It also generates graphs for normalized coverage across transcript. Usually, the middle is much higher than the ends. With this RNA-seq, the ends are actually slightly higher.

                  It's really bizarre. I've never seen anything like this.

                  Comment


                  • #10
                    Actually Illumina has excellent tech support. I suggest you phone or email them. There may be something obvious happening, but it isn't apparent to me.

                    --
                    Phillip

                    Comment

                    Latest Articles

                    Collapse

                    • seqadmin
                      Best Practices for Single-Cell Sequencing Analysis
                      by seqadmin



                      While isolating and preparing single cells for sequencing was historically the bottleneck, recent technological advancements have shifted the challenge to data analysis. This highlights the rapidly evolving nature of single-cell sequencing. The inherent complexity of single-cell analysis has intensified with the surge in data volume and the incorporation of diverse and more complex datasets. This article explores the challenges in analysis, examines common pitfalls, offers...
                      06-06-2024, 07:15 AM
                    • seqadmin
                      Latest Developments in Precision Medicine
                      by seqadmin



                      Technological advances have led to drastic improvements in the field of precision medicine, enabling more personalized approaches to treatment. This article explores four leading groups that are overcoming many of the challenges of genomic profiling and precision medicine through their innovative platforms and technologies.

                      Somatic Genomics
                      “We have such a tremendous amount of genetic diversity that exists within each of us, and not just between us as individuals,”...
                      05-24-2024, 01:16 PM

                    ad_right_rmr

                    Collapse

                    News

                    Collapse

                    Topics Statistics Last Post
                    Started by seqadmin, Today, 07:23 AM
                    0 responses
                    8 views
                    0 likes
                    Last Post seqadmin  
                    Started by seqadmin, 06-17-2024, 06:54 AM
                    0 responses
                    12 views
                    0 likes
                    Last Post seqadmin  
                    Started by seqadmin, 06-14-2024, 07:24 AM
                    0 responses
                    24 views
                    0 likes
                    Last Post seqadmin  
                    Started by seqadmin, 06-13-2024, 08:58 AM
                    0 responses
                    18 views
                    0 likes
                    Last Post seqadmin  
                    Working...
                    X