Genotyping-by-Sequencing Protocol for Genetic Diversity

Genotyping-by-sequencing was performed following the method described by Qi et al. (2018) (link) using the enzyme combination of PstI/MspI. Briefly, 250 ng of DNA from each sample was double-digested with PstI and MspI, followed by the barcode adapter ligation at the PstI site and a common Y-adapter at the MspI site. Unligated adapters were removed by the recovery system of the improved magnetic bead. Recovered fragments with a length of 300–700 bp were PCR-amplified and tested for density using Qubit2.0 to ensure density values greater than 5 ng/μl. The libraries were subsequently sequenced using the Illumina HiSeq X Ten, PE150 platform at OE Biotech Co., Ltd., Qingdao, China.

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Mu X.Y., Wu Y.M., Shen X.L., Tong L., Lei F.W., Xia X.F, & Ning Y. (2022). Genomic Data Reveals Profound Genetic Structure and Multiple Glacial Refugia in Lonicera oblata (Caprifoliaceae), a Threatened Montane Shrub Endemic to North China. Frontiers in Plant Science, 13, 832559.

Publication 2022

HiSeq X Ten, PE150 platform

Enzyme Ligation

Corresponding Organization :

Other organizations : Beijing Forestry University, Beijing Museum of Natural History, Institute of Wetland Research, Chinese Academy of Forestry

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Variable analysis

independent variables

Enzyme combination of Pst I/Msp I used for double-digestion of DNA samples

dependent variables

Sequencing of the libraries using the Illumina HiSeq X Ten, PE150 platform

control variables

Amount of DNA used for double-digestion (250 ng per sample)
Size range of recovered fragments (300-700 bp)
Density of amplified libraries (greater than 5 ng/μl)

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

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