Methods and compositions for deconvoluting partition barcodes
Methods are provided of assessing whether multiple differently-barcoded primers occur within the same partition.
1. A method of detecting the presence or absence of multiple barcodes in a partition, the method comprising,
forming partitions comprising
forward primers comprising a barcode and a capture sequence complementary to a 3′ sequence, or reverse complement thereof, of a sample target nucleic acid, wherein different partitions contain different forward primers comprising different barcode sequences unique to the partitions where the barcodes reside compared to barcodes present in other partitions and wherein in at least one partition the forward primers comprise copies of first and second forward primers comprising different barcode sequences,
the sample target nucleic acid,
multiple copies of a partition ID tag oligonucleotide having a combination of sequences unique to the partition in which the partition ID tag oligonucleotide resides and_comprising a 3′ end sequence that comprises a reverse complement of the capture sequence and a 5′ sequence comprising a variable partition ID tag sequence, wherein the partition ID tag oligonucleotide is a DNA oligonucleotide;
in the partitions, hybridizing the capture sequence of at least some copies of the first and the second forward primers to different copies of the partition ID tag oligonucleotide to form hybridized products, and
hybridizing the capture sequence of other copies of the first and the second forward primers to the sample target nucleic acid;
performing polymerase chain reaction amplification on the hybridized products to form amplicons, wherein at least some amplicons are formed from a first forward primer and the partition ID tag oligonucleotide and at least some amplicons are formed from a second forward primer and the partition ID tag oligonucleotide; and
sequencing the amplicons, wherein if different forward primers form amplicons with the same variable partition ID tag sequence in a partition, the different forward primers are from the same partition, wherein
the partition ID tag oligonucleotide comprises a double-stranded variable partition ID tag sequence and one or two single-stranded 3′ ends comprising the reverse complement of the capture sequence.
2. The method of claim 1 , wherein the forward primers, the partition ID tag oligonucleotide, or both further comprise a universal adaptor sequence.
3. The method of claim 1 , wherein the performing also amplifies the sample target nucleic acid with the forward primers and sample target reverse primers to form an amplification product and the sequencing comprises sequencing the amplification products.
4. The method of claim 1 , wherein in the partition forming step the forward primers are linked to a bead.
5. The method of claim 4 , wherein the forward primers are cleaved from the bead before the amplifying.
6. The method of claim 1 , wherein multiple copies of partition ID tag oligonucleotides are linked to each other.
7. The method of claim 6 , wherein the multiple copies are linked to each other through a bead.
8. The method of claim 7 , wherein the bead is linked to forward primers and partition ID tag oligonucleotides and the bead comprises at least 10 times the number of the forward primers compared to the partition ID tag oligonucleotides linked to the bead.
9. The method of claim 6 , wherein during partition forming the forward primers and the partition ID tag oligonucleotides are linked to each other via a cleavable linker and the method further comprises cleaving the cleavable linker before the amplification.
10. The method of claim 1 , wherein the partition ID tag oligonucleotide is single-stranded.
11. The method of claim 1 , wherein the variable partition ID tag sequence is double-stranded and comprises two single-stranded 3′ ends comprising the reverse complement of the capture sequence.
12. The method of claim 1 , wherein during the partition forming step the partition ID tag oligonucleotide is linked to a solid support.
13. The method of claim 1 , wherein the partition ID tag oligonucleotide comprises an affinity tag.
14. The method of claim 13 , wherein the affinity tag is biotin, an antibody, or an aptamer.
15. The method of claim 1 , wherein the partitions are droplets.
16. The method of claim 1 , wherein contents of the partitions comprising the hybridized product are combined before the amplifying.
17. The method of claim 1 , wherein sample target reverse primers are present in the partitions.
18. The method of claim 1 , wherein sample target reverse primers are introduced after contents of the partitions are combined.
19. The method of claim 1 , wherein the amplification occurs while the hybridized products are within the partitions.
20. The method of claim 1 , wherein the partition ID tag oligonucleotide is 4-50 nucleotides in length.
21. The method of claim 1 , wherein the variable partition ID tag sequence is double-stranded and comprises only one single-stranded 3′ end comprising the reverse complement of the capture sequence.