A user experiencing periodic 10 bp peaks in RNA-seq data from an Illumina NovaSeq X Plus was informed by a self-identified Illumina employee that this pattern is a known artifact of...

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  "detected_at": "2026-09-23T07:53:20+00:00",
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    "domain": "illumina.com"
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    "topics": [
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    "post_id": "1wnzkwc",
    "summary": "A user experiencing periodic 10 bp peaks in RNA-seq data from an Illumina NovaSeq X Plus was informed by a self-identified Illumina employee that this pattern is a known artifact of tagmentation-based library preps.",
    "category": "reliability",
    "comments": [
      {
        "url": "https://www.reddit.com/r/bioinformatics/comments/1wnzkwc/comment/pbizk9g/",
        "depth": 0,
        "score": 21,
        "author": "chewgl",
        "excerpt": "Bead or surface based tagmentation will give you those results. The periodicity of DNA's (cDNA in this case) helical turn is 10 nucleotides, and so the insert lengths will tend towards multiples of that turn (I work at Illumina, have seen such data before in tagmentation-based library prep).",
        "posted_at": "2026-09-23T08:20:50.000Z",
        "author_url": "https://www.reddit.com/user/chewgl/"
      },
      {
        "url": "https://www.reddit.com/r/bioinformatics/comments/1wnzkwc/comment/pbiwwpg/",
        "depth": 0,
        "score": 5,
        "author": "videek",
        "excerpt": "My wild guess on presented intormation alone - PCR bias creating duplicates. But you skipped deaecibing multiple steps before going directly to the alignment.\n\n What do the tapestation results show? How deeply was it sequenced? What peeprocessing steps did you perform? Physiologically intact liver tissue?",
        "posted_at": "2026-09-23T07:57:17.000Z",
        "author_url": "https://www.reddit.com/user/videek/"
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      {
        "url": "https://www.reddit.com/r/bioinformatics/comments/1wnzkwc/comment/pbiz60h/",
        "depth": 0,
        "score": 5,
        "author": "alasthennars",
        "excerpt": "If your insert size is averaging ~125 bases, 2x150 PE sequencing means you are actually reading the whole insert twice, one from each end. Is that the case if you compare your paired reads? This could result in \"too short\" unmapped reads.\n\n Also, since the facility used polyA enrichment, if the trace you are showing is for the reverse reads, it could be that the weird peaks correspond to different",
        "posted_at": "2026-09-23T08:17:18.000Z",
        "author_url": "https://www.reddit.com/user/alasthennars/"
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      {
        "url": "https://www.reddit.com/r/bioinformatics/comments/1wnzkwc/comment/pbjhxl9/",
        "depth": 0,
        "score": 5,
        "author": "refutalisk",
        "excerpt": "The nextera results in this paper have a similar periodicity. https://pmc.ncbi.nlm.nih.gov/articles/PMC8805253/",
        "posted_at": "2026-09-23T10:52:28.000Z",
        "author_url": "https://www.reddit.com/user/refutalisk/"
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      {
        "url": "https://www.reddit.com/r/bioinformatics/comments/1wnzkwc/comment/pbjibka/",
        "depth": 0,
        "score": 2,
        "author": "pombe",
        "excerpt": "\"too short\" doesn't mean that the read sequences are short in the sense of number of base pairs. It's that the portion of the read that aligns to the reference isnt long enough to be counted.\n\n Did you accidentally align it to the wrong genome?",
        "posted_at": "2026-09-23T10:55:08.000Z",
        "author_url": "https://www.reddit.com/user/pombe/"
      },
      {
        "url": "https://www.reddit.com/r/bioinformatics/comments/1wnzkwc/comment/pbl3e4v/",
        "depth": 0,
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        "author": "MexicaUrbano",
        "excerpt": "Overtagmentation plus very deep sequencing would be my diagnosis. You have no tagmentation step in your protocol so I am going to change my answer to severe RNA degradation plus protein steric hindrance. Notice how much less prevalent the bumps become once you reach a specific size, consistent with a steric process.\n\n The more important point is your insert size is considerably less than 300bp, wh",
        "posted_at": "2026-09-23T15:40:45.000Z",
        "author_url": "https://www.reddit.com/user/MexicaUrbano/"
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    "evidence": [
      "[post] Sequencing: 2x150 PE, NovaSeq X Plus",
      "[comment u/chewgl] Bead or surface based tagmentation will give you those results. The periodicity of DNA's (cDNA in this case) helical turn is 10 nucleotides, and so the insert lengths will tend towards multiples of that turn (I work at Illumina, have seen such data before in tagmentation-based library prep)."
    ],
    "virality": "somewhat_high",
    "post_date": "2026-09-23T07:53:20.000Z",
    "post_kind": "multi_media",
    "post_text": "Hi everyone!\n\nI'm seeing a strange insert size distribution in one of my RNA-seq samples. Instead of a smooth curve, there are regular peaks roughly every 10 bp.\n\nIn the STAR log, about 50% of reads are unmapped as \"too short\", so a large part of the library seems to be very short fragments.\n\nSetup:\n\nLibrary prep: NEBNext Ultra II Directional RNA + Poly(A) mRNA Magnetic Isolation Module (done by a sequencing facility)\n\nSequencing: 2x150 PE, NovaSeq X Plus\n\nSample type: liver tissue\n\nRIN: 8.9\n\nHas anyone seen this pattern in a poly-A library before? Is the data still usable (e.g. counting only the reads that do map), or is this sample a lost cause?\n\nThanks!",
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      "technical question"
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    "post_title": "Weird periodic insert size distribution (~10 bp peaks) in RNA-seq",
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    "source_url": "https://www.reddit.com/r/bioinformatics/comments/1wnzkwc/weird_periodic_insert_size_distribution_10_bp/",
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    "comments_total": 22,
    "total_comments": 22,
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        "name": "New England Biolabs",
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    "comments_included": 6,
    "products_mentioned": [
      "NovaSeq X Plus",
      "Nextera"
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