Reducing PCR bias for NGS library construction

Next-generation sequencing library construction includes many steps: e.g., LD-PCR (long-distance PCR, 2 kb in length), then acoustic shearing to the appropriate size and used in a standard Illumina library preparation (involving end-repair and kination, A-tailing and adapter ligation, followed by additional amplification by PCR) [1]. Library preparations can be susceptible to bias resulting from genomes that contain unusually high or low GC content and approaches have been developed to address these situations through careful selection of polymerases for PCR amplification, thermocycling, conditions and buffers [2]. But also, the thermal cycler used influences the PCR result and improvement is possible.

Steep, fast ramp rates have been shown to be inappropriate for amplification of GC rich targets (above 80 % GC). The “steep thermoprofile does not leave sufficient time above a critical threshold temperature” so that “incomplete denaturation and poor amplification” can be the result [2]. There are multiple options to improve your PCR protocol for obtaining better results, such as addition of chemicals, change of polymerase, etc.

What to consider while NGS library preparation

In this article, we highlight three prospects related to thermal cyclers with a fast heating ramp rate to reduce PCR bias when preparing NGS libraries:

1. Optimization of the PCR protocol: Improvement includes elongation of the initial denaturation time and the denaturation time in every cycle by 6-8 times than given in the protocol [2]. But this elongates the whole PCR process and lapses the most obvious advantage of a fast ramping cycler.

2. Adapt the ramping speed: Some thermal cyclers have the option to program a desired ramping speed. Like this it is easy to soften the heating and cooling to reduce bias during PCR.

3. Play with the denaturation temperature: not only elongation of the denaturation step, but also testing different denaturation temperatures can be beneficial for adequate denaturation of GC rich targets and improve the PCR result. This can be done by either programming a gradient step in the denaturation step (if the cycler only offers 1 gradient option), or by programming a gradient in the denaturation and annealing step (if the cycler offers a 2D-gradient). The advantages of a 2D gradient are obvious. Two steps of the PCR protocol can be optimized in one run, while for a thermal cycler that offers only 1 gradient two PCR runs are necessary.

Successful preparation of your NGS library

Overall, it is possible to use a fast ramping thermal cycler for NGS PCR applications, but a slower ramp rate showed beneficial results for GC-rich targets from the start without optimization effort. When it comes to the thermal cycler itself, we recommend using a slower ramping thermal cycler such as the Mastercycler^® X40 with a max. of 3.3 °C/s heating ramp rate that can still be reduced if necessary.


1. Steven R. Head,1 H. Kiyomi Komori,2 Sarah A. LaMere,2 Thomas Whisenant,2 Filip Van Nieuwerburgh,3 Daniel R. Salomon,2 and Phillip Ordoukhanian. Library construction for next-generation sequencing: Overviews and challenges. Biotechniques. 2014; 56(2):61

2. Aird D, Ross MG, Chen WS, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 2011;12:R18