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[Blog] Bubble Product: Decreasing the Efficiency of Target Enrichment

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Next Generation Sequencing (NGS), particularly short-read NGS using platforms like Illumina and MGI, requires adapter sequences attached to both ends of the DNA library. Consequently, all samples have identical sequences at both ends. These identical sequences can cause a byproduct known as Bubble product during library preparation, leading to various experimental and sequencing challenges. This post will explain how these bubbles form and impact target capture experiments and sequencing.

1. Bubble Formation and Changes in DNA Library Patterns


Bubbles form when all DNA fragments in the library have identical adapter sequences at both ends, and primers are significantly consumed during PCR. As large volumes of primers are consumed, instead of primers binding to the adapter regions, two DNA strands from different fragments anneal to each other.

image_DNA Library Bubble_celemics_blog#1
Figure 1. Structure of Bubble product

Two main factors significantly influence bubble formation:

  1. Amount of input DNA
  2. Number of PCR cycles

DNA quality can also cause bubble formation, acting as a variable in establishing stable experimental conditions. Generally, bubbles are more likely to be formed in enzymatic preparation (EP) kits that simultaneously perform end-repair and A-tailing while cutting large DNA, such as genomic DNA, than library preparation kits that use pre-cut DNA. This is because, in the case of EP kits, the longer the cutting time, the more DNA fragments are produced.


As bubbles form, the intensity of target DNA bands are gradually reduced (Figures 2-1 to 2-3), eventually leading to highly inaccurate size and concentration (Figure 2-4).

Figure 2. DNA library pattern changes after bubble forms

2. Impact of Bubbles


The presence of Bubbles makes it difficult to determine the exact concentration and size of the DNA library. This poses a problem, especially when sequencing must be performed immediately after library preparation, as the inability to accurately ascertain concentration and size can lead to biased data production. Furthermore, for downstream experiments like Target Enrichment with multiplexing of samples, these Bubbles can result in significant ratio discrepancies between samples, causing bias in experimental results.

3. Preventing Bubbles


Through our research, we have identified the following major factors that can lead to bubble formation in library preparation procedure:

  1. Input DNA amount
  2. DNA fragmentation time (for enzymatic library preparation kits)
  3. Pre-PCR cycle adjustment
  4. Post-PCR cycle adjustment

We aim to assist customers using Celemics’ Library Preparation and Target Enrichment kits in finding the optimal conditions to prevent Bubble formation for their experiments. Please contact us to achieve successful results under optimal experimental conditions.