Conjoined synthetic polynucleotides ladders

A composition comprising:

• a target polynucleotide;
• and
• multiple different conjoined synthetic polynucleotides for use as ladders in a sequencing assay, each conjoined synthetic polynucleotide comprising:
• a first number of copies of a first synthetic polynucleotide;
• and
• a second number of copies of a second synthetic polynucleotide, different from the first number of copies, wherein the first number of copies and the second number of copies define a predetermined ratio of numbers of copies of the first and second synthetic polynucleotides that the conjoined polynucleotide is designed to have for use as a ladder in a sequencing assay;
• and
• one or more linkers physically linking the copies of the first synthetic polynucleotide and the copies of the second polynucleotide in the conjoined synthetic polynucleotide to mitigate stoichiometric variation of the predetermined ratio due to pipetting error during sample preparation for the sequencing assay;
• wherein the first polynucleotide and the second polynucleotide have different nucleotide sequences;
• wherein at least two of the multiple different conjoined synthetic polynucleotides are at different concentrations in the composition.

A conjoined synthetic polynucleotide designed for use as a ladder in a sequencing assay, the conjoined synthetic polynucleotide comprising:

• a first number of copies of a first synthetic polynucleotide;
• a second number of copies of a second synthetic polynucleotide, different from the first number of copies, wherein the first number of copies and the second number of copies define a predetermined ratio of numbers of copies of the first and second synthetic polynucleotides that the conjoined polynucleotide is designed to have for use as the ladder in the sequencing assay;
• and
• one or more linkers physically linking the copies of the first synthetic polynucleotide and the copies of the second polynucleotide in the conjoined synthetic polynucleotide to mitigate stoichiometric variation of the predetermined ratio due to pipetting error during sample preparation for the sequencing assay;
• wherein the first polynucleotide and the second polynucleotide have different nucleotide sequences.