Optimal enzymes for amplifying sequencing libraries

摘要: PCR amplification introduces bias into Illumina sequencing libraries1. Although amplification-free library preparation solves this, micrograms of starting material are usually required. Most researchers follow standard protocols using Phusion polymerase, which has processivity and fidelity advantages over most polymerases. Yet for genomics applications, our demands on DNA systems often surpass their specification. Thermostable polymerases such as used to amplify mixtures fragments, albeit with variable efficiency. Typically, (G+C)-neutral fragments amplified higher efficiency than extremely (G+C)-rich or (A+T)-rich fragments. The accumulation these slight differences in multiple cycles results profound bias. There have been reports alternative construction2, 3, 4, but infrequent, comprehensive analyses lacking. To reduce bias, we investigated many thermostable alternate reaction conditions adapter-ligated sequencing. We expect this comparison be relevant other applications that involve simultaneous complex fragment mixtures.