Abstract

BackgroundNovel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits.ResultsWe compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits’ fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection.ConclusionsThe enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Highlights

  • Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings

  • In this study we examined the performance of four whole genome sequencing (WGS) library preparation kits that employ enzymatic DNA fragmentation in comparison to the “tagmentation”based Illumina DNA library preparation kit

  • We tested the performance of four WGS library prep kits employing enzymatic fragmentation in comparison to the transposition-based Nextera DNA flex kit

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Summary

Introduction

Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Illumina technology has come to dominate short read generation sequencing (NGS), offering cost-effective high precision data for a wide variety of applications such as whole genome sequencing (WGS), metagenomics and transcriptomics. Library preparation is an essential process preceding sequencing itself, and comprises several aspects that affect the efficiency of WGS. It typically involves the following main steps: fragmentation of the input DNA, endrepair and A-tailing of the DNA fragments, ligation of indexed sequencing adapters and optional amplification of the ligated products. One or more cleanup steps are necessary in between steps to purify the DNA reaction products of reagents from the previous reaction

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