Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Problems with adaptor ligation - NEBNExtUltra II

    Hello to you all,

    I want to perform DNA seq and I am having issues in the step of ligating the adaptors to the sequences.

    To give you more details: We want to find the different virus detected in an individual. For that purpose, we eliminated the host genome using DNAses and RNAses on the lysate, and then inactivated the nucleases and performed an additional RNA/DNA isolation step with GeneJet Viral DNA/RNA isolation kit. Then, using the Ovation system by Tekan, samples were RT to dsDNA. Then DNA was purified again using Qiagen columns.

    At this point, samples were quantified using nanodrop (most of them were at around 100ng/ul with good ratios), and Qubit with equally good results. Size profiles were assessed with Bioanalyzer HS DNA chip, and profiles looked ok.

    We performed then library prep with NEBNExtUltra II starting from 300ng and then a qPCR to measure number of sequences containing adaptors, and there was barely any detection. We checked using a Bioanalyzer the profiles again and there was indeed no shift of the samples, what should be observed after the ligation with the adapters.

    We repeated the protocol with two samples in parallel, one from our cohort, another a commercial DNA used as a control. The control sample worked perfectly. There was no detection of adapters on our sample.

    Has anyone been on a similar situation? Any idea on what may be the issue here?

    Thanks,

    J

  • #2
    NEB adapters are short hairpins so you may not see a shift in size, better to check it after PCR. Without PCR step qPCR will not work as they are not full length adapters and lack qPCR primer binding region.

    Comment


    • #3
      Thanks nucacidhunter,
      We run a "control" library and we did see a shift on size for this one, although in this case, this was genomic DNA non-derived from virus. Do you think that could be the determining factor?
      Thanks again,

      J

      Comment


      • #4
        I wonder if you could provide the exact name and product # of Ovation kit used. Ovation RNA Amplification System V2 results in single stranded DNA that cannot be used for double stranded adapter ligation. It also primes cDNA synthesis from polyA. If your viral RNA lacks polyA then I wonder where the final product has come from. If you could post Bioanalyzer trace of amplified cDNA and possibly your starting material I can have a look.

        Comment


        • #5
          Many thanks again! The exact kit that used was 7102-08-FG (previously 7102-08) OVATION RNA-SEQ V2 08 .

          According to what we found online, this should produce dsDNA molecules that are not dependent of polyA.

          Waiting on your answer and many thanks for you help,

          J

          Comment


          • #6
            Successful ligation with NEB kit requires fragment end-prep (blunting, 5'phosphorylation and 3' adenylation). SPIA uses RNA/DNA hybrid primers that will persist in at least one end of fragments and presence of ribonucleotide will prevent end-prep so they need to be fragmented before end-prep to remove ribonucleotide. If you have used fragmented SPIA amplified cDNA, I would recommend contacting Tecan Techsupport.

            Comment


            • #7
              Will check that out. Many thanks

              Comment


              • #8
                How does the Ovation RNA seq v2 kit generate double-stranded cDNA instead of single-stranded cDNA??!!


                I am trying to use the Ovation RNA seq V2 kit from Nugen or Tecan to amplify low-input RNA (about 1ng).
                (https://lifesciences.tecan.com/ovation-low-input-rna-seq-kit-v2?p=tab--1).

                The Protocol and workflow of the SPIA amplification process show linear amplification of cDNA, which is single-stranded (As per the schematic provided by them). However, the kit claims it generates double-stranded cDNA, which is not shown in the figure or explained how it is done!!!

                Can somebody please help me understand this?

                How does the Ovation RNA seq v2 kit generate double-stranded cDNA instead of single-stranded cDNA??!!

                Nugen/ tecan suggests using their library preparation kits (for Illumina) which can utilize only dsDNA templates but not single-stranded.
                They seem to work well, but I don't understand how the dsDNA is generated in the amplification step (RNA Seq V2 kit).
                Click image for larger version

Name:	BMC_SPIA.gif
Views:	145
Size:	60.8 KB
ID:	324041
                Please help.

                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