Unconfigured Ad

Collapse
X
 
  • Filter
  • Time
  • Show
Clear All
new posts
  • ZAAB
    Member
    • Apr 2011
    • 18

    50+% of my HiSEQ reads are 3' primer (custom primer used)

    Hi All:
    We've run 7 lanes of Illumina sequencing with the HiSEQ.
    We're sequencing the middle of a PCR product...
    We're using a custom sequencing primer to avoid the common 5' end of the PCR product.
    In many of our lanes, we 'see' about 50% of the reads being the 3' end Illumina adapter (barcode side)...

    If we were just using the common Illumina adapter primer (hybridizing to the 5' end), I'd say I have a huge Illumina primer-dimer issue...
    BUT I am using a custom primer...

    So how does this happen?
    Did I get my forward, non-5'-phosphate PCR primer filled in when A-tailing (without, ligation of adapters has bad efficiency (Separate issue)), and then Illumina primers ligated to it...
    IS this possible>?

    OR Is there regular Illumina sequencing primer contamination in my runs, and the Illumina primer dimer is being sequenced...?

    Or?

    Thanks in advance.
  • NextGenSeq
    Senior Member
    • Apr 2009
    • 482

    #2
    Did you synthesize your adapters with phosphothioate bonds in between the ends of the last two 3' bases?

    This prevents exonuclease digestion and adapter artifacts.

    Comment

    • ZAAB
      Member
      • Apr 2011
      • 18

      #3
      My PCR primers for generating the construct BEFORE illumina adapter ligation are just regular primers.
      I used the Illumina DNA sample prep kit, so the primers were Illumina, and hence, should have had that O-bond.

      Comment

      • NextGenSeq
        Senior Member
        • Apr 2009
        • 482

        #4
        I would redesign my PCR primers and cut down on the concentration. If you still get primer artifacts gel purify before adapter ligation.

        I assume you purified by some method before ligating the adapters? If not there will be PCR primers with your product.

        Also, don't design the primers with an A at the end since the T overhang from the adaptor might bind to it.

        Comment

        Latest Articles

        Collapse

        • SEQadmin2
          Advanced Sequencing Platforms Tackle Neuroscience’s Toughest Genomics Problems
          by SEQadmin2



          Genomics studies in neuroscience face a special challenge due to the brain’s complexity and scarcity of samples. Mapping changes in cell type and state using conventional next-generation sequencing methods remains challenging. Advances in technologies like single-cell sequencing, spatial transcriptomics, and long-read sequencing have opened the door to deeper studies of the brain and diseases like Alzheimer’s, amyotrophic lateral sclerosis (ALS), and schizophrenia.
          ...
          07-09-2026, 11:10 AM
        • SEQadmin2
          Cancer Drug Resistance: The Lingering Barrier to Rising Survival
          by SEQadmin2



          Cancer survival rates have significantly increased in the last few decades in the United States, reaching a combined 70% 5-year survival rate by 2021. Behind this number, there are years of research to find new therapies, drug targets, and early detection methods. But there is one core challenge that keeps slowing down these advances, and it’s about drug resistance.

          There is no single reason why many patients don’t respond to treatment as expected. Cancer is...
          07-08-2026, 05:17 AM
        • GATTACAT
          Reply to Nine Things a Sample Prep Scientist Thinks About Before Sequencing
          by GATTACAT
          Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
          07-01-2026, 11:43 AM

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by SEQadmin2, 07-13-2026, 10:26 AM
        0 responses
        18 views
        0 reactions
        Last Post SEQadmin2  
        Started by SEQadmin2, 07-09-2026, 10:04 AM
        0 responses
        30 views
        0 reactions
        Last Post SEQadmin2  
        Started by SEQadmin2, 07-08-2026, 10:08 AM
        0 responses
        16 views
        0 reactions
        Last Post SEQadmin2  
        Started by SEQadmin2, 07-07-2026, 11:05 AM
        0 responses
        34 views
        0 reactions
        Last Post SEQadmin2  
        Working...