51 - Implementation of a New Automated Sample Quality Control Tool in a Whole Exome Sequencing Workflow

Abstract

Objectives: The German Cancer Research Center (DKFZ) is one of the largest biomedical research institutions in Germany. The High Throughput Sequencing Unit of the DKFZ Genomics and Proteomics Core Facility provides sequencing services for multiple applications. This project demonstrates the use of an automated electrophoresis system as a quality control (QC) tool in a whole exome sequencing workflow. Mandatory for the experimental success of whole exome sequencing is the quality of the incoming genomic DNA (gDNA) material and the DNA samples at various stages of the library preparation workflow. Methodology: Exome libraries were prepared according to the Agilent Low Input Sure-SelectXT Human All Exon v5 protocol from FFPE tumor tissue samples. The libraries were equimolar pooled. Each pool was sequenced on two lanes using the Illumina HiSeq 4000 System with 100 bp paired end sequencing. To ensure success quality control was verified with an Agilent 4200 TapeStation system of the received gDNA samples and during the library preparation. Results: Intermediate QC steps were taken throughout the protocol to monitor library preparation for sequencing, such as evaluation of DNA after fragmentation, analysis of adapter-ligated and amplified DNA, and lastly, qualification of the final library. The initial QC of incoming gDNA was determined based on the DNA integrity number (DIN). All samples had a low DNA integrity, what is usual for DNA extracted from FPPE material. Due to the low quality of the DNA material, a modified fragmentation protocol was used. Modification of the fragmentation enabled to obtain meaningful sequencing results. Conclusion: Quality control is an important part of NGS workflows, library preparation protocols recommend quantification and qualification of the DNA samples at various stages. The increasing sample throughput creates a need for automation especially in a core facility where many precious samples are proceeded with time pressure. The implementation of the automated electrophoresis system in the whole exome sequencing workflow enabled to increase the efficiency of the workflow and ensure good sequencing results.