Methods and kits for identifying and adjusting for bias in sequencing of polynucleotide samples

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.