Conjoined synthetic polynucleotides

A conjoined synthetic polynucleotide for use in identifying polymerase chain reaction amplification bias between different alleles of a human gene, the conjoined synthetic polynucleotide comprising a single contiguous polynucleic acid that comprises:

• (a) a first polynucleotide comprising:
• a first primer binding site,
• a reference associated portion of a reference allele of the human gene,
• a first synthetic portion, and
• a reverse complement of a second primer binding site;
• (b) a second polynucleotide in a 1:1 ratio in the conjoined synthetic polynucleotide with the first polynucleotide, the second polynucleotide comprising:
• a third primer binding site having the same sequence as the first primer binding site,
• a variant associated portion of a variant allele of the human gene;
• a second synthetic portion;
• and
• a reverse complement of a fourth primer binding site having the same sequence as the reverse complement of the second primer binding site;
• and
• (c) a synthetic linker physically linking the first polynucleotide and the second polynucleotide to mitigate stoichiometric variation of the 1:1 ratio due to pipetting error.

A conjoined synthetic polynucleotide comprising a plurality of pairs of first and second polynucleotides in a single contiguous polynucleic acid, each pair of the plurality of pairs comprising:

• (a) a first polynucleotide comprising:
• a first primer binding site,
• a reference associated portion of a reference allele of a human gene,
• a first synthetic portion, and
• a reverse complement of a second primer binding site;
• (b) a second polynucleotide in a 1:1 ratio in the conjoined synthetic polynucleotide with the first polynucleotide, wherein the second polynucleotide comprises:
• a third primer binding site having the same sequence as the first primer binding site,
• a variant associated portion of a variant allele of the human gene;
• a second synthetic portion;
• and
• a reverse complement of a fourth primer binding site having the same sequence as the reverse complement of the second primer binding site;
• and
• (c) a synthetic linker physically linking the first polynucleotide and the second polynucleotide to mitigate stoichiometric variation of the 1:1 ratio due to pipetting error.

A method of determining an allele fraction of a variant allele of a human gene in a sample, the method comprising:

• (a) adding a known quantity of a conjoined synthetic polynucleotide standard to a sample comprising a variant allele and a reference allele of the human gene,
• wherein the conjoined synthetic polynucleotide standard comprises a single contiguous polynucleic acid comprising:
• (i) a first polynucleotide comprising:
• a first primer binding site,
• a reference associated portion of a reference allele of the human gene,
• a first synthetic portion, and
• a reverse complement of a second primer binding site;
• (ii) a second polynucleotide in a 1:1 ratio in the conjoined synthetic polynucleotide with the first polynucleotide, wherein the second polynucleotide comprises:
• a third primer binding site having the same sequence as the first primer binding site,
• a variant associated portion of a variant allele of the human gene;
• a second synthetic portion;
• and
• a reverse complement of a fourth primer binding site having the same sequence as the reverse complement of the second primer binding site;
• and
• (iii) a synthetic linker physically linking the first polynucleotide and the second polynucleotide to mitigate stoichiometric variation of the 1:1 ratio due to pipetting error;
• wherein the first primer binding site and the second primer binding site are both naturally occurring portions of the human gene that are conserved in the reference allele and the variant allele;
• (b) amplifying the reference allele, the variant allele, the first polynucleotide, and the second polynucleotide using a first pair of primers that are complementary to the first primer binding site and the second primer binding site, respectively, to produce amplified polynucleotides;
• (c) sequencing the amplified polynucleotides to generate sequencing reads associated with the reference allele, the variant allele, the first polynucleotide, and the second polynucleotide;
• (d) determining amplification bias of the reference allele and the variant allele by measuring a deviation from the 1:1 ratio between sequence reads derived from the first polynucleotide and sequence reads derived from the second polynucleotide;
• and
• (e) determining the allele fraction of the variant allele in the sample based on the amplification bias and the sequence reads derived from the reference allele and the variant allele.