Novel assay for phasing of distant genomic loci with zygosity resolution via long-read sequencing hybrid data analysis

A method of phasing (i) at least one distant single nucleotide polymorphism of interest and (ii) short tandem repeats and/or at least one exonic single nucleotide polymorphism of interest within the same target gene locus of nucleic acids isolated from a biological sample, the method comprising:

• a. performing an amplification step comprising contacting genomic DNA isolated from said biological sample comprising the target gene locus with a first set of oligonucleotide primers to produce a first amplification product comprising the at least one distant single nucleotide polymorphism of interest and at least one exonic reference single nucleotide polymorphism in the same target gene locus;
• b. performing a reverse transcription and amplification step comprising contacting mRNA isolated from said biological sample comprising the target gene locus with a second set of oligonucleotide primers to produce a second amplification product comprising the short tandem repeats and/or the at least one exonic single nucleotide polymorphism of interest and said at least one exonic reference single nucleotide polymorphism;
• c. determining nucleic acid sequences of the first and the second amplification product;
• d. aligning the nucleic acid sequences of the first and the second amplification product determined in step (c) based on a position of the at least one exonic reference single nucleotide polymorphism;
• and
• e. determining a haplotype of (i) the at least one distant single nucleotide polymorphism of interest and (ii) the short tandem repeats and/or the at least one exonic single nucleotide polymorphism of interest in the sample.