Detection of sequences uniquely associated with a DNA target region
The disclosed embodiments concern methods for determining sequences of interest using targeted unique molecular index (TUMI) sequences that are uniquely associated with individual polynucleotide fragments in plants, such as those present in a transgenic event, a site-specific mutation or a wild type variant. System, apparatus, and computer program products are also provided for determining a sequence of interest implementing the methods disclosed.
1. A method for detecting a target nucleotide region of interest in a plant genome, wherein the plant genome is from wheat, potato, cotton, apple, alfalfa or canola, by directional nucleotide sequencing, the method comprising:
providing a first polynucleotide fragment comprising a first polynucleotide PCR primer sequence specific to the target nucleotide region of interest, a sequencing primer binding site sequence, and a polynucleotide sequence region uniquely associated with the target nucleotide region of interest, wherein the sequencing primer binding site is positioned between the polynucleotide sequence uniquely associated with the target nucleotide region of interest and the first polynucleotide PCR primer sequence specific to the target nucleotide region of interest;
providing a second polynucleotide fragment comprising a second polynucleotide PCR primer sequence specific to the target nucleotide region of interest;
performing a PCR to create one or more amplicons comprising the sequencing primer binding site sequence, the polynucleotide sequence uniquely associated with the target nucleotide region of interest, and the target nucleotide region of interest;
sequencing the one or more amplicons using the sequencing primer in a direction away from the target nucleotide region of interest so that the polynucleotide sequence uniquely associated with the target nucleotide region of interest is sequenced and the target nucleotide region of interest in the one or more amplicons is not sequenced; and
detecting the target nucleotide region in the plant genome based on the sequence information of the polynucleotide sequence uniquely associated with the target nucleotide region of interest.
2. The method of claim 1 , wherein the target nucleotide region of interest is a transgene.
3. The method of claim 2 , wherein the transgene confers herbicide tolerance.
4. The method of claim 2 , wherein the transgene confers disease resistance.
5. The method of claim 2 , wherein the transgene confers insect resistance.
6. The method of claim 1 , wherein sequencing adapters are attached to the ends of the one or more amplicons through a second PCR reaction performed prior to the sequencing reaction.
7. The method of claim 1 , wherein one or more sample indices are present on the one or more amplicons, wherein the one or more sample indices are added to at least one of the first polynucleotide fragment and the second polynucleotide fragment, or are added to the amplicons comprising the sequencing primer binding site sequence, the polynucleotide sequence uniquely associated with the target nucleotide region of interest, and the target nucleotide region of interest by a subsequent round of PCR.
8. The method of claim 1 , wherein at least one of the first polynucleotide PCR primer sequence specific to the target nucleotide region of interest and the second polynucleotide PCR primer sequence specific to the target nucleotide region of interest hybridizes to a sequence that spans a border sequence between genomic DNA and the target nucleotide region of interest.
9. The method of claim 1 , wherein at least one of the first polynucleotide PCR primer sequence specific to the target nucleotide region of interest and the second polynucleotide PCR primer sequence specific to the target nucleotide region of interest hybridizes to a sequence within the target nucleotide region of interest.
10. The method of claim 1 , wherein the target nucleotide region of interest comprises a single nucleotide polymorphism.
11. The method of claim 1 , wherein the target nucleotide region of interest comprises an introduced site-specific genome modification.
12. The method of claim 1 , wherein the target nucleotide region of interest comprises an allelic variation or a haplotype.
13. The method of claim 1 , wherein adapter oligonucleotides that are complementary to flow-cell anchor oligonucleotides are added to the one or more amplicons by a second round of PCR.
14. The method of claim 13 , wherein prior to the second round of PCR, primer dimers are reduced by a purification or selection step.
15. The method of claim 1 , wherein the PCR is quantitative.
16. The method of claim 1 , wherein more than one polynucleotide sequence region uniquely associated with a nucleotide region of interest is utilized, and each such polynucleotide sequence region uniquely associated with a target nucleotide region of interest has a Hamming distance of at least 4 from each other such sequence.
17. The method of claim 1 , further comprising the detection of a genomic marker region comprising the steps of:
during the PCR reaction, also creating one or more amplicons each comprising a genomic marker region, wherein an additional sequencing primer binding site is positioned on the amplicon proximal to the genomic marker region; and
sequencing the one or more amplicons using an additional sequencing primer, wherein the additional sequencing primer binding site is positioned such that the additional sequencing primer will direct sequencing to occur toward and through the genomic marker region.
18. The method of claim 17 , wherein the genomic marker region comprises one or more SNP markers.
19. The method of claim 1 , wherein the plant genome is from wheat.
20. The method of claim 1 , wherein the plant genome is from canola.
21. The method of claim 1 , wherein the target nucleotide region of interest is a CRISPR modified trait.