| Abstract [eng] |
Haplotyping – the process of determining co-inherited alleles occurring on a single strand of DNA, has a high priority for diploid genome characterization and its association with the phenotype. Currently, the most widely used next generation sequencing workflows generate short sequencing reads and represent genotypes – genetic variations in the unknown molecular origin. Due to short reads, genetic variations farther than the contig length are not resolved directly and require additional in silico methods. Because indirect haplotyping is based on statistical genotype data comparison and most accurately resolve short haplotypes, molecular or direct haplotyping methods are required for whole genome analysis, as well as for rare and de novo mutation research. The aim of this work was to develop a system capable of resolving haplotype information in parallel with whole genome sequencing. Oligonucleotides immobilized on polystyrene spheres with random primers and bead barcodes were used to achieve virtual space separation between DNA molecules. The conditions for next generation sequencing library preparation method were optimized - the efficiency of oligonucleotide-tethered nucleotide incorporation and extension, the optimal number of particles, amount of genomic DNA and oligonucleotide-tethered nucleotides in the reaction, as well as non-specific reactions and efficiency of side product removal. The optimized next generation sequencing library preparation method was applied to human whole genome sequencing and the haplotyping efficiency was evaluated in three different systems. |
