Compositions and methods comprising asymmetric barcoding
In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided.
1. A method of identifying a copy number variation of a genetic locus in a sample, said method comprising:
(a) subjecting sense strand circular polynucleotides and antisense strand circular polynucleotides to a rolling circle amplification reaction using (i) sense strand primers comprising a sense strand identifying barcode and (ii) antisense strand primers comprising an antisense strand identifying barcode, wherein said rolling circle amplification reaction yields barcoded concatemers, each comprising a junction sequence formed at a circularization junction of a circular polynucleotide and multiple repeats of said circular polynucleotide sequence;
(b) subjecting said barcoded concatemers to a primer extension reaction using a first set and a second set of auxiliary primers to generate amplicons, wherein the first set of auxiliary primers hybridize to a barcoded concatemer of a sense strand and the second set of auxiliary primers hybridize to a barcoded concatemer of an antisense strand, individual primers of the first and the second set of auxiliary primers are distinctly barcoded with respect to other members of the same set, and wherein individual amplicons generated are distinguishable from each other by a unique combination of (i) the strand identifying barcode at one end and (ii) a distinct auxiliary barcode at the other end;
(c) sequencing the amplicons or derivatives thereof to produce sequencing reads;
(d) identifying one or more distinct polynucleotides comprising the genetic locus as originating from distinct single-stranded target polynucleotides via (i) a distinct junction sequence or (ii) the strand identifying barcode; and
(e) identifying a copy number variation of the genetic locus when the number of distinct polynucleotides comprising the genetic locus, when compared to a number of distinct polynucleotides comprising a reference region, indicates a copy number variation of the genetic locus.
2. The method of claim 1 , wherein prior to (d), sequencing reads are grouped into amplicon families, wherein an amplicon family comprises sequencing reads having the same strand identifying barcode, the same auxiliary barcode, and the same junction sequence.
3. The method of claim 1 , wherein sense strand circular polynucleotides and antisense strand circular polynucleotides are generated by (i) forming single-stranded polynucleotides from double-stranded polynucleotides, and (ii) subjecting said single-stranded polynucleotides to a ligation reaction.
4. The method of claim 1 , wherein sense strand circular polynucleotides and antisense strand circular polynucleotides are generated by (i) subjecting double-stranded polynucleotides to a ligation reaction to yield double-stranded, circular polynucleotides, and (ii) forming single-stranded, circular polynucleotides from said double-stranded, circular polynucleotides.
5. The method of claim 1 , wherein barcoded concatemers in (b) comprise two or more repeats of the circular polynucleotide sequence.
6. The method of claim 1 , wherein said sense strand primers and/or said anti-sense primers comprise a gene specific sequence.
7. The method of claim 1 , wherein primers of said first set and/or said second set of auxiliary primers comprise gene specific sequences.
8. The method of claim 1 , wherein said sense strand primer and/or said anti-sense strand primer further comprise a tag sequence, a sequencing primer binding sequence, or both.
9. The method of claim 1 , wherein primers of said first set and/or said second set of auxiliary primers further comprise a tag sequence, a sequencing primer binding sequence, or both.
10. The method of claim 1 , wherein said copy number variation is correlated with a disease.
11. The method of claim 1 , wherein said sample comprises cell-free polynucleotides.
12. The method of claim 1 , wherein said sample comprises polynucleotides from tumor cells.
13. The method of claim 1 , wherein said sample comprises fragmented genomic DNA.
14. The method of claim 1 , wherein said sample comprises less than 50 ng of polynucleotides.
15. The method of claim 1 , wherein said barcoded concatemer comprises at least two copies of said circular polynucleotide.