High density sequencing and multiplexed priming
Disclosed herein, inter alia, are methods and compositions for sequencing a plurality of template nucleic acids.
1. A method of sequencing different populations of polynucleotides immobilized on a solid support, wherein the solid support is a multiwell container, said method comprising:
contacting a first population of polynucleotides annealed to a first sequencing primer with a first sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator and monitoring the sequential incorporation of complementary nucleotides to generate a first sequencing read, wherein the reversible terminator is removed prior to the introduction of the next complementary nucleotide, wherein the first sequencing primer comprises SEQ ID NO: 1;
contacting a second population of polynucleotides annealed to a second sequencing primer with a second sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator and monitoring the sequential incorporation of complementary nucleotides to generate a second sequencing read, wherein the reversible terminator is removed prior to the introduction of the next complementary nucleotide; and
contacting a third population of polynucleotides annealed to a third sequencing primer with a third sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator and monitoring the sequential incorporation of complementary nucleotides to generate a third sequencing read, wherein the reversible terminator is removed prior to the introduction of the next complementary nucleotide;
wherein the first sequencing primer, second sequencing primer, and third sequencing primer are different.
2. The method of claim 1 , comprising sequencing a plurality of different populations of polynucleotides within overlapping optically resolvable features.
3. The method of claim 2 , wherein the first population of polynucleotides, the second population of polynucleotides, and the third population of polynucleotides are not substantially complementary.
4. The method of claim 1 , wherein monitoring the sequential incorporation of complementary nucleotides comprises a sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-binding process.
5. The method of claim 1 , wherein monitoring the sequential incorporation of complementary nucleotides comprises incorporating one or more modified nucleotides into the sequencing primer with a polymerase to create an extension strand, and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby generating one or more sequencing reads.
6. The method of claim 1 , wherein monitoring the sequential incorporation of complementary nucleotides comprises
incorporating one or more modified nucleotides into the first sequencing primer with a polymerase to create an extension strand, and detecting the one or more incorporated nucleotides in a first optically resolvable feature;
incorporating one or more modified nucleotides into the second sequencing primer with a polymerase to create an extension strand, and detecting the one or more incorporated nucleotides in a second optically resolvable feature; and
incorporating one or more modified nucleotides into the third sequencing primer with a polymerase to create an extension strand, and detecting the one or more incorporated nucleotides in a third optically resolvable feature; wherein the first, second, and third optically resolvable features overlap.
7. The method of claim 1 , comprising generating about 500 million (M) to about 3×10 11 sequencing reads.
8. The method of claim 1 , wherein the multiwell container solid comprises a plurality of oligonucleotides immobilized to a polymer.
9. The method of claim 1 , wherein the multiwell container comprises a plurality of particles.
10. The method of claim 1 , wherein the multiwell container comprises a plurality of oligonucleotides at a density of about 100 oligonucleotides per μm 2 to about 1,000,000 oligonucleotides per μm 2 .
11. The method of claim 1 , wherein each template polynucleotide is not reseeded, reamplified, or both reseeded and reamplified after generating each sequencing read.
12. The method of claim 2 , wherein the method generates a total number of sequencing reads greater than the number of optically resolvable features on the multiwell container.
13. The method of claim 2 , wherein the method generates a total number of sequencing reads greater than 100% of the optically resolvable features on the multiwell container.
14. The method of claim 1 , wherein the first population of polynucleotides, the second population of polynucleotides, and the third population of polynucleotides each comprise a platform primer binding sequence, wherein said platform primer binding sequence is the same.
15. The method of claim 1 , wherein the first population of polynucleotides comprises a first and a second sequencing primer binding sequence, and the second population of polynucleotides comprises a first and a third sequencing primer binding sequence.
16. The method of claim 1 , wherein the first population of polynucleotides comprises a first and a second sequencing primer binding sequence, and the second population of polynucleotides comprises a third and a fourth sequencing primer binding sequence.
17. The method of claim 6 , wherein the optically resolvable features overlap by at least 25%.
18. The method of claim 6 , wherein the optically resolvable features overlap by between at least 25% to 75%.
19. The method of claim 1 , comprising monitoring the sequential incorporation of 5 or more complementary nucleotides prior to contacting a population of polynucleotides with a different sequencing solution.
20. The method of claim 1 , comprising monitoring the sequential incorporation of 5 to 100 complementary nucleotides prior to contacting a population of polynucleotides with a different sequencing solution.
21. A method of sequencing different polynucleotides immobilized on a solid support, wherein the solid support is a multiwell container, said method comprising:
contacting a first polynucleotide annealed to a first sequencing primer with a first sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator, and incorporating one or more modified nucleotides into the first sequencing primer with a polymerase to create a first extension strand, and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the first extension strand, thereby generating a first sequencing read, wherein the first sequencing primer comprises SEQ ID NO: 1;
contacting a second polynucleotide annealed to a second sequencing primer with a second sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator and incorporating one or more modified nucleotides into the second sequencing primer with a polymerase to create a second extension strand, and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the second extension strand, thereby generating a second sequencing read; and
contacting a third polynucleotide annealed to a third sequencing primer with a third sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator and incorporating one or more modified nucleotides into the third sequencing primer with a polymerase to create a third extension strand, and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the third extension strand, thereby generating a third sequencing read;
wherein the first sequencing primer, second sequencing primer, and third sequencing primer are different.