Abstract
The use of next-generation sequencing (NGS) has been instrumental in advancing biological research and clinical diagnostics. To fully utilize the power of NGS, complete, uniform coverage of the entire genome is required. In this study, we identified the primary sources of bias observed in sequence coverage across AT-rich regions of the human genome with existing amplification-free DNA library preparation methods. We have found evidence that a major source of bias is the inefficient processing of AT-rich DNA in end repair and 3′ A-tailing, causing under-representation of extremely AT-rich regions. We have employed immobilized DNA modifying enzymes to catalyze end repair and 3′ A-tailing reactions, to notably reduce the GC bias observed with existing library construction methods.
Highlights
Next-generation sequencing (NGS) has revolutionized both biology and medical diagnosis[1,2]
The negative control (NC) reactions showed the positions of 54 nt and 51 nt strands. (c) Degradation study using a pair of 3′ recessed synthetic DNA substrates labeled with 5′ FAM
PKT treatment of 47-AT at 37 °C resulted in detection of degradation species that are smaller than the expected 51 nt product from primer extension whereas the negative control (NC) reactions showed the positions of 47 nt and 51 nt strands
Summary
Next-generation sequencing (NGS) has revolutionized both biology and medical diagnosis[1,2]. A typical protocol of amplification-free library preparation for the Illumina platform comprises fragmentation, end repair (blunting and 5′ phosphorylation), 3′ A-tailing and adaptor ligation[11]. We hypothesized that due to DNA thermal breathing AT-rich DNA fragments are subjected to nuclease-mediated degradation and other events leading to under-representation To prove this hypothesis, we devised an enzyme immobilization strategy to remove enzymes and to circumvent the requirement for high temperature incubation by physically removing the enzymes (Fig. 1). Enzymes covalently conjugated to magnetic beads successfully catalyzed DNA end-polishing steps in library preparation for the Illumina sequencing platform at low temperature, leading to significantly increased coverage of high AT-content regions of human DNA libraries
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