High throughput SNP genotyping with two mini-sequencing assays.
Two mini-sequencing methods, FP-TDI (template-directed dye-terminator incorporation with fluorescence-polarization) and MassArray (matrix assisted laser desorption ionization time of flight detection mass spectrometry), were optimized. A numeric standard was introduced to evaluate the SNP scoring quality of FP-TDI assay, thus made the optimization work easier. At the same time, using multi-PCR technology, 8-plex genotyping of MassArray assay was successfully carried out, some softwares were developed and the data process of MassArray was highly automated. Then these two methods were applied to high throughput SNP genotyping, the accuracy, efficiency and robustness were compared. The result shows FP-TDI is more sensitive to the concentration of SNPprimer and PCR product, as well as extension cycles, the SNPprimer length of FP-TDI should be 24 to 30 bp long, whereas MassArray assay prefers to be as short as only 16 bp. Altogether 6440 SNP sites of human chromosome 3 were genotyped in a sample of 90 individuals, 4792 sites by FP-TDI assay and 1648 sites by MassArray assay, the success rates of FP-TDI and MassArray were 67.7% and 93.6% respectively. The throughput of MassArray was higher than FP-TDI, and the cost of MassArray was lower, MassArray was more suitable for high throughput SNP genotyping.
Luo
Chunqing
C
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 101300, China.
Deng
Libin
L
Zeng
Changqing
C
D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D015233:Models, Statistical; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods
D017931:DNA Primers; D004247:DNA