Big dye terminator version

The genomic DNA was extracted using a GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific, USA) as per the product instruction. The SNP genotyping work was performed using an improved multiplex ligation detection reaction (iMLDR) technique developed by Genesky Biotechnologies Inc. (Shanghai, China). A multiplex PCR-ligase detection reaction method was used in the iMLDR. For each SNP, the alleles were distinguished by different fluorescent labels of allele-specific oligonucleotide probe pairs. Different SNPs were further distinguished by different extended lengths at the 3′end. Two negative controls were set: one with double-distilled water as template and the other with DNA sample without primers while keeping all other conditions the same in one plate. Duplicate tests were designed and the results were consistent. Genotyping call rate is 100%. A random sample accounting for ~ 5% (n = 40) of the total DNA samples was directly sequenced using Big Dye-terminator version 3.1 and an ABI3730XL automated sequencer (Applied Biosystems) to confirm the results of iMLDR.

Candidate variants were confirmed by Sanger sequencing when DNA was available. DNA was amplified by polymerase chain reaction using Roche FastStart PCR Master Mix (Roche Diagnostics Corp) and sequenced with Applied Biosystems BigDye terminator version 3.1 sequencing chemistry in an ABI3730XL genetic analyzer as per manufacturer’s instructions (Applied Biosystems). Primers are available upon request. The sequences were analysed using Sequencher software version 4.2 (Gene Codes).