Methods and kits for identifying and adjusting for bias in sequencing of polynucleotide samples
View Patent ↗Disclosed are methods for determining one or more nucleotides at one or more nucleotide positions of a polynucleotide sample, the polynucleotide sample comprising heterogeneous polynucleotides having different nucleotides at the nucleotide positions. The disclosed methods may be utilized to control for sequencing bias during sequencing of the polynucleotide sample. Suitable samples may include patient samples for use in diagnosing, prognosing, and treating the patient.
1. A method for detecting a cytosine nucleotide or a thymine nucleotide at different nucleotide positions of a polynucleotide sample, the polynucleotide sample comprising heterogeneous polynucleotides having a cytosine nucleotide or a thymidine nucleotide at the different nucleotide positions, the method comprising:
(a) sequencing in the reaction mixture a target region of the polynucleotide sample that spans the different nucleotide positions;
(b) sequencing in the same reaction mixture as an internal control a set of polynucleotide fragments comprising a polynucleotide sequence corresponding to the target region of the polynucleotide sample, the polynucleotide fragments further comprising a nucleotide tag for distinguishing the sequenced polynucleotide fragments from the sequenced target region of the polynucleotide sample, the set of polynucleotide fragments comprising all possible polynucleotide fragments having different combinations of a cytosine or a thymidine at the different nucleotide positions, wherein the polynucleotide sample and each of the polynucleotide fragments are present in equimolar amounts in the same reaction mixture; and
(c) determining that a cytosine nucleotide or a thymine nucleotide is present at the different nucleotide positions of the polynucleotide sample.
2. The method of claim 1 , wherein performing sequencing comprises performing deep sequencing.
3. The method of claim 1 , further comprising determining depth of sequencing required to detect a cytosine nucleotide or a thymidine nucleotide at the different nucleotide positions.
4. The method of claim 1 , further comprising determining sequencing bias for a cytosine nucleotide versus a thymidine nucleotide at the different nucleotide positions of the polynucleotide sample.
5. The method of claim 1 , further comprising determining a false-discovery rate for a cytosine nucleotide versus a thymine nucleotide at the different nucleotide positions of the polynucleotide sample.
6. The method of claim 1 , wherein the polynucleotide sample is obtained from genomic DNA.
7. The method of claim 6 , wherein the polynucleotide sample is prepared by treating the genomic DNA with a reagent that selectively modifies non-methylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues.
8. The method of claim 1 , further comprising calculating a ratio of the heterogeneous polynucleotides of the polynucleotide sample having a cytosine nucleotide at the different nucleotide positions to the heterogeneous polynucleotides of the polynucleotide sample having a thymidine nucleotide at the different nucleotide positions.
9. The method of claim 1 , wherein the nucleotide tag comprises a nucleotide sequence of at least about 2 nucleotides that is not present in the target region of the polynucleotide sample and sequencing the set of polynucleotides fragments comprises utilizing a primer that specifically hybridizes to a polynucleotide comprising the nucleotide tag.