title: The Role of Quality Control in Targeted Next-generation Sequencing Library Preparation.
abstract: Next-generation sequencing (NGS) is getting routinely used in the diagnosis of hereditary diseases, such as human cardiomyopathies. Hence, it is of utter importance to secure high quality sequencing data, enabling the identification of disease-relevant mutations or the conclusion of negative test results. During the process of sample preparation, each protocol for target enrichment library preparation has its own requirements for quality control (QC); however, there is little evidence on the actual impact of these guidelines on resulting data quality. In this study, we analyzed the impact of QC during the diverse library preparation steps of Agilent SureSelect XT target enrichment and Illumina sequencing. We quantified the parameters for a cohort of around 600 samples, which include starting amount of DNA, amount of sheared DNA, smallest and largest fragment size of the starting DNA; amount of DNA after the pre-PCR, and smallest and largest fragment size of the resulting DNA; as well as the amount of the final library, the corresponding smallest and largest fragment size, and the number of detected variants. Intriguingly, there is a high tolerance for variations in all QC steps, meaning that within the boundaries proposed in the current study, a considerable variance at each step of QC can be well tolerated without compromising NGS quality.
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany.
authors.forename: Rouven
authors.initials: R
authors.lastname: Nietsch
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Haas
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany.
authors.forename: Alan
authors.initials: A
authors.lastname: Lai
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Oehler
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mester
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Frese
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany.
authors.forename: Farbod
authors.initials: F
authors.lastname: Sedaghat-Hamedani
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany.
authors.forename: Elham
authors.initials: E
authors.lastname: Kayvanpour
authors.affiliation: Chair for Clinical Bioinformatics, Medical Faculty, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Keller
authors.affiliation: Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany. Electronic address: benjamin.meder@med.uni-heidelberg.de.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Meder
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D006801:Humans; D011786:Quality Control; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards
chemical_list: D004247:DNA
keywords: Library preparation; Next-generation sequencing; Quality control; Sequence variants; Target enrichment