Reconditioning PCR for removal of PCR bubbles in Illumina librarys

Abstract

This protocol describes how to remove partly single-stranded PCR products ("PCR bubbles") from Illumina libraries. For more information about this phenomenon please see this explanation from Illumina. For metabarcoding libraries, it can be hard to estimate the optimal input template or the perfect amount of PCR cycles and therefore overamplification happens frequently. PCR bubbles cannot be quantified reliably with fluorometric-based methods and may look different on different capillary electrophoresis devices.

PCR bubbles can lead to failed sequencing runs due to over- or underloading the flowcell.

Steps

Quality control

Perform a quality control of your library. PCR bubbles may look different depending on the method used for quality control. See below and example of the Fragment Analyzer, Bioanalyzer and an agarose gel.

Example result of the Agielnt fragment analyzer for a library that contains PCR bubbles. The desired peak is at 443 bp, the shoulder to the right of it is the PCR bubble.

The same library on the Agilent bioanalyzer. The shoulder is moved further to the right, also the relationship of library to PCR bubble changed significantly.

PCR reactions of the same library visualized on 1% agarose. Notice the faint band above the actual amplicon.

Library concentration (optional)

Concentrate your library by reducing the volume down to 100µL . We usually do this with a spin-column based protocol, although this can be performed with magnetic beads as well.

Note

Please see: or

Reconditioning PCR

Fill the concentration of your library and the project name into the Excel spreadsheet. The suggested master mix for the reconditioning PCR will be calculated accordingly. We usually go for 1250ng of template input, however, this can be adjusted if necessary. master mix

Note

You can download the Excel spreadsheet here:https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.x54v9p9ypg3e/Mastermix%20calculator%20reconditioning%20PCR.xlsx

Perform the PCR with 4 reactions of 50µL .

PCR clean-up

Pool the 4 PCR reactions.

Perform a column-based PCR clean-up to exchange the buffer. This can also be done with magnetic beads. Elute the DNA in 100µL .

Guanidine-based DNA extraction with silica-coated beads or silica spin columns

Size-selection

Perform a size selection with a ratio of 0.7x to remove residual primer dimers. Elute the final library in 50µL .

PCR cleanup and size selection with magnetic beads

Perform final quality control

Perform a final quality control. Quantify the library concentration with a fluorometric-based method and perform quality control via electrophoresis. The shoulder should be gone and the library should be good for sequencing.

Example result of the Agielnt fragment analyzer for a library after the removal of PCR bubbles.

The same library on the Agilent bioanalyzer after the removal of PCR bubbles.

Citation