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The Role of Quality Control in Targeted Next-generation Sequencing Library Preparation.

Authors
  • Nietsch, Rouven1
  • Haas, Jan2
  • Lai, Alan1
  • Oehler, Daniel2
  • Mester, Stefan2
  • Frese, Karen S2
  • Sedaghat-Hamedani, Farbod2
  • Kayvanpour, Elham2
  • Keller, Andreas3
  • Meder, Benjamin4
  • 1 Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany. , (Germany)
  • 2 Institute for Cardiomyopathies, Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; German Centre for Cardiovascular Research (DZHK), Heidelberg/Mannheim, Germany. , (Germany)
  • 3 Chair for Clinical Bioinformatics, Medical Faculty, Saarland University, 66123 Saarbrücken, Germany. , (Germany)
  • 4 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: [email protected] , (Germany)
Type
Published Article
Journal
Genomics, proteomics & bioinformatics
Publication Date
Aug 01, 2016
Volume
14
Issue
4
Pages
200–206
Identifiers
DOI: 10.1016/j.gpb.2016.04.007
PMID: 27475404
Source
Medline
Keywords
License
Unknown

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.

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