Determining abundance of target polynucleotides

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

• combining the sample with multiple synthetic polynucleotides, each synthetic polynucleotide of the multiple synthetic polynucleotides comprising:
• (i) target-associated portions that are also present in the target polynucleotide and that can be used to amplify the synthetic polynucleotide along with the target polynucleotide using a first pair of primers, and
• (ii) a barcode that can be used to distinguish among otherwise identical synthetic polynucleotides;
• amplifying the target polynucleotide and the multiple synthetic polynucleotides using the first pair of primers to produce amplified polynucleotides;
• sequencing the amplified polynucleotides to generate:
• (i) sequence reads derived from the target polynucleotide, and (ii) sequence reads derived from the multiple synthetic polynucleotides;
• and
• determining the abundance of the target polynucleotide in the sample based on:
• (i) the sequence reads derived from the target polynucleotide, and (ii) the sequence reads derived from the multiple synthetic polynucleotides.

A method of determining polynucleotide abundance and/or sequencing error, the method comprising:

• (a) combining a sample with multiple synthetic polynucleotides, each of the multiple synthetic polynucleotides comprising:
• (i) a first primer binding site that is also present in a target polynucleotide of the sample;
• and
• (ii) a barcode that can be used to distinguish among otherwise identical synthetic polynucleotides;
• and
• (iii) a reverse complement of a second primer binding site, wherein the reverse complement of the second primer binding site is also present in the target polynucleotide;
• amplifying the target polynucleotide and the synthetic polynucleotide using a first primer that is complementary to the first primer binding site and a second primer that is complementary to the second primer binding site to generate amplified polynucleotides;
• sequencing the amplified polynucleotides to generate:
• (i) sequence reads derived from the target polynucleotide, and (ii) sequence reads derived from the multiple synthetic polynucleotides;
• and
• identifying sequence reads corresponding to particular synthetic polynucleotides of the multiple synthetic polynucleotides using the barcodes;
• and
• (b) determining the abundance of the target polynucleotide in the sample based on:
• (i) the sequence reads derived from the target polynucleotide, and (ii) the sequencing reads derived from the particular synthetic polynucleotides;
• (c) determining a sequencing error rate based on identifying differences between:
• (i) sequences of the sequence reads corresponding to the particular synthetic polynucleotides and (ii) known sequences of the particular synthetic polynucleotides;
• or
• (d) determining the abundance of the target polynucleotide in the sample based on:
• (i) the sequence reads derived from the target polynucleotide, and (ii) the sequencing reads derived from the particular synthetic polynucleotides;
• and determining a sequencing error rate based on identifying differences between:
• (i) sequences of the sequence reads corresponding to the particular synthetic polynucleotides and (ii) known sequences of the particular synthetic polynucleotides.

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

• (a) combining the sample with at least one synthetic polynucleotide, wherein the at least one synthetic polynucleotide comprises:
• (i) a target-associated portion having sequence identity to the target polynucleotide to enable co-amplification of the target polynucleotide and the at least one synthetic polynucleotide;
• and
• (ii) a variation portion comprising a varied nucleotide sequence of a corresponding window of the target polynucleotide, wherein the varied nucleotide sequence has a different sequence than the sequence of the corresponding window of the target polynucleotide to remove sequence homology between the target polynucleotide and the at least one synthetic polynucleotide;
• (b) co-amplifying the target polynucleotide and the at least one synthetic polynucleotide in a single reaction to generate amplified polynucleotides;
• (c) sequencing the amplified polynucleotides to generate sequence reads;
• and
• (d) determining the abundance of the target polynucleotide based on a comparison of the sequence reads derived from the target polynucleotide against the sequence reads derived from the at least one synthetic polynucleotide.