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- W2948332208 abstract "Long-range PCR (LR-PCR) is used to enrich the target regions of the genome. This study aimed to establish the pipeline of targeted gene sequencing using LR-PCR and massively parallel sequencing (MPS).The 14-kb-long MEFV gene, including the entire coding exons, was selected as a target gene and amplified using LR-PCR. The evaluated analytical factors were as follows: LR-PCR conditions, three types of post-PCR cleanup methods, and two types of MPS library preparation methods.With regard to LR-PCR conditions, Tks Gflex DNA polymerase at 7-min (30-s/kb) annealing/extension with 100-ng genomic DNA input had the highest yield. Regarding post-PCR purification methods, the magnetic beads-based method had high recovery and purity. In the MPS library preparation methods, the ligation-based method had a higher base coverage in the target (94.58%), uniformity of base coverage (99.95%), and target bases with no strand bias (97.40%). The exonic variants determined by Sanger sequencing were detected by both ligation- and transposon-based methods.Various analytical factors were evaluated, and the pipeline of targeted gene sequencing using LR-PCR and MPS was established. These data can enable the optimization of targeted gene sequencing using LR-PCR and MPS in the clinical laboratory." @default.
- W2948332208 created "2019-06-14" @default.
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- W2948332208 date "2019-08-01" @default.
- W2948332208 modified "2023-09-24" @default.
- W2948332208 title "Evaluation of analytical factors associated with targeted MEFV gene sequencing using long-range PCR/massively parallel sequencing of whole blood DNA for molecular diagnosis of Familial Mediterranean fever" @default.
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- W2948332208 doi "https://doi.org/10.1016/j.cca.2019.06.001" @default.
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