Bubble products in sequencing libraries: causes, identification, and workflow recommendations

A bubble product is a heteroduplex composed of partially homologous library fragments. These heteroduplex molecules contain double-stranded complementary adapter sequences flanking single-stranded noncomplementary inserts. The result is a partially open, bubble-like conformation in the middle of the fragment. When bubble products migrate on a Bioanalyzer trace (or an equivalent instrument), the partially open conformation slows the migration through the gel matrix. This slower migration shows as an artificial peak that typically migrates around twice the size in base pairs of the expected library peak.

In the figure below, the main product migrates around 300 bp. The additional peak at approximately 600 bp is comprised of bubble product.

Figure 1: Library showing bubble product in addition to the primary product

What causes bubble products to form?

In the early stages of PCR (Figure 2a) primers are in excess, and each template anneals to the appropriate primer. Annealed primers extend and generate complementary strands.

In later amplification cycles (Figure 2b), the concentration of library increases. Eventually, the desired library-primer hybridization may be outcompeted by library-library hybridization. When two library strands anneal, they can have noncomplementary inserts that result in library with hybridized adapters flanking an unhybridized insert region.

Bubble product may be the result of too much input, or too many cycles of PCR.

Figure 2: Formation of bubble product: primers are two shades of green and template is blue or purple insert

How to limit bubble product formation?

• Use the recommended amount of nucleic acid input, and quantify input using a fluorometric method for accuracy.

• Follow the appropriate library preparation protocol regarding the recommended number of PCR cycles.

Can libraries with a bubble product peak be sequenced?

Yes, libraries showing a bubble product peak can be sequenced. Both primary and secondary bubble products will be denatured into single-stranded DNA prior to flow cell binding, which will remove the artificial “bubble.” After denaturation, the single-stranded DNA from both the bubble product peak and the expected peak will bind to the flow cell and sequence normally.

Libraries showing a bubble product peak must be quantified by qPCR, using the expected peak size for the size adjustment calculation. Both the primary product and bubble product will be quantified because libraries are denatured into single-stranded DNA for qPCR.

Using a fluorometric-based method to quantify the library when bubble product is present will result in inaccurate quantification. Fluorometric-based methods only quantify double-stranded DNA, while bubble product consists of both double-stranded DNA and single-stranded DNA. Fluorometric quantification will underestimate the library concentrations, which can negatively impact run performance.

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