期刊
BIOTECHNIQUES
卷 55, 期 2, 页码 61-67出版社
FUTURE SCI LTD
DOI: 10.2144/000114063
关键词
droplet digital PCR; absolute quantification; next-generation sequencing; massively parallel sequencing; library preparation; quality control; size determination
资金
- Ellison Medical Foundation [AG-NS-0577-09]
- National Institute of Environmental Health Sciences [R01ES019319]
- U.S. Department of Defense [W81XWH-10-1-0563]
Due to the high cost of failed runs and suboptimal data yields, quantification and determination of fragment size range are crucial steps in the library preparation process for massively parallel sequencing (or next-generation sequencing). Current library quality control methods commonly involve quantification using real-time quantitative PCR and size determination using gel or capillary electrophoresis. These methods are laborious and subject to a number of significant limitations that can make library calibration unreliable. Herein, we propose and test an alternative method for quality control of sequencing libraries using droplet digital PCR (ddPCR). By exploiting a correlation we have discovered between droplet fluorescence and amplicon size, we achieve the joint quantification and size determination of target DNA with a single ddPCR assay. We demonstrate the accuracy and precision of applying this method to the preparation of sequencing libraries.
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