Methods of sequencing using conjoined synthetic polynucleotide ladders

A method of determining abundance of a target polynucleotide in a sample, the method comprising:

• (a) adding a known quantity of a conjoined synthetic polynucleotide to the sample, wherein the conjoined synthetic polynucleotide comprises:
• 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, wherein the first polynucleotide and the second polynucleotide have different nucleotide sequences;
• 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 sequencing;
• (b) fragmenting the conjoined synthetic polynucleotide to produce fragmented synthetic polynucleotides;
• (c) co-amplifying the target polynucleotide and the fragmented synthetic polynucleotides in a single reaction to generate amplified products;
• (d) sequencing the amplified products to generate sequence reads;
• and
• (e) estimating quantitative error in the generated sequence reads, the estimating comprising comparing:
• (i) differential abundances of the first synthetic polynucleotide and the second synthetic polynucleotide determined using the generated sequence reads derived from the first and second synthetic polynucleotides, respectively, to (ii) the predetermined ratio of the numbers of copies of the first and second synthetic polynucleotides;
• and
• (f) determining the abundance of the target polynucleotide in the sample using sequence reads derived from the target polynucleotide and the quantitative error in the generated sequence reads.

A method of determining abundance of a target polynucleotide in a sample, the method comprising:

• (a) adding a known quantity of a conjoined synthetic polynucleotide to the sample, wherein the conjoined synthetic polynucleotide comprises:
• 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, wherein the first polynucleotide and the second polynucleotide have different nucleotide sequences;
• 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 sequencing,
• wherein the one or more linkers comprise an endonuclease site and are positioned in the conjoined synthetic polynucleotide such that a linker of the linkers is located between each of the copies of the first and second synthetic polynucleotides;
• wherein the first polynucleotide and/or the second polynucleotide comprise:
• (a) a synthetic portion and (b) a target-associated portion that is complementary to the target polynucleotide;
• (b) liberating the copies of the first and the second synthetic polynucleotides from the conjoined synthetic polynucleotide using an endonuclease that is cognate to the endonuclease site to produce liberated copies of the first and the second synthetic polynucleotides;
• (c) co-amplifying the target polynucleotide and the liberated copies of the first and the second synthetic polynucleotides in a single reaction to generate amplified products;
• (d) sequencing the amplified products to generate sequence reads;
• and
• (e) estimating quantitative error in the generated sequence reads, the estimating comprising comparing:
• (i) differential abundances of the first synthetic polynucleotide and the second synthetic polynucleotide determined using the generated sequence reads derived from the first and second synthetic polynucleotides, respectively, to (ii) the predetermined ratio of the numbers of copies of the first and second synthetic polynucleotides;
• and
• (f) determining the abundance of the target polynucleotide in the sample using sequence reads derived from the target polynucleotide and the quantitative error in the generated sequence reads.

A method of determining abundance of a target polynucleotide in a sample, the method comprising:

• (a) adding a known quantity of a conjoined synthetic polynucleotide to the sample, wherein the conjoined synthetic polynucleotide comprises:
• 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, wherein the first polynucleotide and the second polynucleotide have different nucleotide sequences;
• (b) after (a), fragmenting polynucleotides of the sample using a next-generation sequences (NGS) polynucleotide fragmentation reagent to produce fragmented polynucleotides;
• (c) sequencing the fragmented polynucleotides to generate sequence reads;
• and
• (d) determining the abundance of the target polynucleotide in the sample using sequence reads derived from the target polynucleotide, sequence reads derived from the first synthetic polynucleotide, sequence reads derived from the second synthetic polynucleotide, and the predetermined ratio.