Sanger Sequencing Variant Verification

Sanger sequencing was performed as previously described to verify the variants identified by targeted NGS [13 (link)]. Briefly, forward and reverse PCR primers were designed to amplify the fragments covering the variant sites. PCR products were purified with shrimp alkaline phosphatase and exonuclease and then directly sequenced on an ABI 3500xL Dx Genetic Analyzer (Applied Biosystems, Foster City, USA). Co-segregation analysis was performed in the families with identified mutations.

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Liu Z.J., Lin H.X., Wei Q., Zhang Q.J., Chen C.X., Tao Q.Q., Liu G.L., Ni W., Gitler A.D., Li H.F, & Wu Z.Y. (2019). Genetic Spectrum and Variability in Chinese Patients with Amyotrophic Lateral Sclerosis. Aging and Disease, 10(6), 1199-1206.

Publication 2019

ABI 3500xL Dx Genetic Analyzer

Alkaline phosphatase Exonuclease Genetic Mutations Primers

Corresponding Organization :

Other organizations : Second Affiliated Hospital of Zhejiang University, First Affiliated Hospital of Fujian Medical University, Fujian Medical University, Stanford University

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Variable analysis

independent variables

Forward and reverse PCR primers designed to amplify the fragments covering the variant sites

dependent variables

Variants identified by targeted NGS
Co-segregation of identified mutations in families

control variables

PCR products purified with shrimp alkaline phosphatase and exonuclease
Directly sequenced on an ABI 3500xL Dx Genetic Analyzer (Applied Biosystems, Foster City, USA)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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