Ultralow Coverage Sequencing Is the Most Accurate Library Quantification Method Prior to Exome Sequencing

Abstract

Accurate DNA library quantification is very important in post-pooling captured exome sequencing. Underrepresented libraries will need additional sequencing, which takes extra time and money, whereas overexpressed DNA libraries can lead to the generation of more data than required, which leads to the waste of sequence capacity and a reduced number of samples per batch. There is a number of methods available to quantify DNA libraries prior to sequencings, such as UV absorption, use of intercalating dyes, capillary electrophoresis, 5’-hydrolysis probes (TaqMan©) coupled with quantitative PCR (qPCR) or droplet digital PCR. But there is no gold standard for the quantification of DNA libraries. This study compares common library quantification methods, including LabChip (PerkinElmer Inc., MA, USA), Qubit 3.0 (Thermo Fisher Scientific, MA, USA), several qPCR approaches, and ultralow coverage sequencing on Illumina MiSeq platform (with and without insert size correction). DNA libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA). To compare the above-mentioned approaches, cost, time, and quantification accuracy were assessed in our study. Quantification methods involving the use of Qubit and MiSeq were found to be better than qPCR and LabChip approaches at predicting the final library concentration. It was also revealed that MiSeq with insert size correction was the most accurate method for library quantification prior to exome sequencing. This method allows for correction shifts in the ratio due to enrichment. Ultralow coverage sequencing on the Illumina MiSeq platform is the most accurate method of library quantification prior to pooling and post-pooling exome enrichment.