Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.

Louise Aigrain,Michael A Quail,Yong Gu

doi:10.1186/s12864-016-2757-4

Abstract

BackgroundThe emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency.ResultsWe compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits.ConclusionWe describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2757-4) contains supplementary material, which is available to authorized users.

Highlights

The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries
We developed an assay based on droplet digital polymerase chain reaction (PCR) technology to measure the amount of DNA remaining after each steps of a protocol, as well as the percentage of fragment bearing adaptors at their ends after the ligation step, or P5/P7 primers after amplification [6]
DNA library preparation kits for Illumina sequencing We tested 9 kits listed in Table 1 following the protocol recommended in each manual but keeping the ratio of beads during the clean-up steps, the PCR reagents and settings for the amplification step identical between kits in order to allow a direct comparison between the droplet digital PCR (ddPCR) results

Summary

Introduction

The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being cost and minimal bias, yield and time efficiency. In contrast with qPCR, ddPCR doesn’t require the use of any standards to calculate the absolute number of specific molecules in a sample [4, 7,8,9,10] This allows the quantification of the overall yield, as normally done with qPCR, and of the yield of some critical intermediate steps such as the adaptor ligation [11,12,13,14]

Methods

Results

Discussion

Conclusion