High density sequencing and multiplexed priming
Disclosed herein, inter alia, are methods and compositions for sequencing a plurality of template nucleic acids. In an aspect is provided a method of sequencing an overlapping amplification cluster, a method of sequencing different polynucleotide immobilized on a solid support, and a method of sequencing three or more populations of polynucleotides on a solid support.
1 . A method of sequencing an overlapping amplification cluster, said method comprising:
contacting a solid support with a first polynucleotide comprising a first sequencing primer binding sequence, a second polynucleotide comprising a second sequencing primer binding sequence, and a third polynucleotide comprising a third sequencing primer binding sequence, wherein each of said polynucleotides and sequencing primer binding sequences are different and distinct;
hybridizing an amplification primer binding sequence of the first polynucleotide to a first oligonucleotide sequence attached to the solid support; hybridizing an amplification primer binding sequence of the second polynucleotide to a second oligonucleotide sequence attached to the solid support; hybridizing an amplification primer binding sequence of the third polynucleotide to a third oligonucleotide sequence attached to the solid support; wherein each of said amplification primer binding sequences are the same, and each of said oligonucleotide sequences are different;
amplifying the polynucleotides thereby forming an overlapping amplification cluster; and
sequentially sequencing said overlapping amplification cluster by hybridizing a first sequencing primer to the first sequencing primer binding sequence, or a complement thereof, and generating a first sequencing read comprising 10 or more nucleotide bases; after generating the first sequencing read, hybridizing a second sequencing primer to the second sequencing primer binding sequence, or a complement thereof, and generating a second sequencing read comprising 10 or more nucleotide bases; after generating the second sequencing read, hybridizing an extendable third sequencing primer to the third sequencing primer binding sequence, or a complement thereof, and generating a third sequencing read comprising 10 or more nucleotide bases; wherein
sequencing comprises contacting each hybridized sequencing primer with a sequencing solution comprising one or more modified nucleotides comprising a reversible terminator, and monitoring the sequential incorporation of said one or more modified nucleotides to generate a sequencing read, wherein the reversible terminator is removed prior to the introduction of the next modified nucleotide, and wherein the reversible terminator of each modified nucleotide is cleaved using the same cleaving agent cleaved by the same cleaving agent.
2 . The method of claim 1 , wherein amplifying comprises bridge polymerase chain reaction (bPCR) amplification, solid-phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, emulsion PCR, or combinations thereof.
3 . The method of claim 1 , wherein sequencing comprises incorporating 10 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 modified nucleotides in a first optically resolvable feature;
incorporating 10 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 modified nucleotides in a second optically resolvable feature; and
incorporating 10 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 modified nucleotides in a third optically resolvable feature, wherein the first, second, and third optically resolvable features overlap.
4 . The method of claim 3 , wherein the method further comprises incorporating a dideoxy nucleotide triphosphate (ddNTP) into the first extension strand prior to hybridizing the second sequencing primer, and wherein the method further comprises incorporating a ddNTP into the second extension strand prior to hybridizing the third sequencing primer.
5 . The method of claim 3 , wherein the optically resolvable features overlap by at least 25%, at least 50%, or at least 75%, and wherein said optically resolvable feature comprises an area of about 0.5 μm 2 to about 1.5 μm 2 .
6 . The method of claim 1 , comprising generating about 500 million (M) to about 3×10 11 sequencing reads.
7 . A method of sequencing different polynucleotides immobilized on a solid support, 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 monitoring the sequential incorporation of 10 or more of said modified nucleotides into the first sequencing primer to generate a first sequencing read, wherein the reversible terminator is removed prior to the introduction of the next modified nucleotide; and after generating the first sequencing read,
hybridizing a second sequencing primer to a second polynucleotide and contacting the second sequencing primer with a second sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator and monitoring the sequential incorporation of 10 or more of said modified nucleotides into the second sequencing primer to generate a second sequencing read, wherein the reversible terminator is removed prior to the introduction of the next modified nucleotide; and after generating the second sequencing read,
hybridizing a third sequencing primer to a third polynucleotide and contacting the third sequencing primer with a third sequencing solution comprising a plurality of modified nucleotides comprising a reversible terminator and monitoring the sequential incorporation of 10 or more of said modified nucleotides into the third sequencing primer to generate a third sequencing read, wherein the reversible terminator is removed prior to the introduction of the next modified nucleotide;
wherein the first sequencing primer, second sequencing primer, and third sequencing primer each comprise different sequences, and the first polynucleotide, second polynucleotide, and third polynucleotide are distinct and comprise different sequences; and wherein the modified nucleotides of the first sequencing solution, second sequencing solution, and third sequencing solution each comprise a reversible terminator capable of being cleaved by the same cleaving agent.
8 . The method of claim 7 , wherein said solid support is a multiwell container.
9 . The method of claim 7 , comprising sequencing a plurality of different polynucleotides within overlapping optically resolvable features.
10 . The method of claim 7 , wherein monitoring the sequential incorporation of complementary nucleotides comprises a sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-binding process.
11 . The method of claim 7 , wherein monitoring the sequential incorporation of modified nucleotides comprises
incorporating 10 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 modified nucleotides in a first optically resolvable feature;
incorporating 10 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 modified nucleotides in a second optically resolvable feature; and
incorporating 10 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 modified nucleotides in a third optically resolvable feature; wherein the first, second, and third optically resolvable features overlap.
12 . The method of claim 7 , comprising generating about 500 million (M) to about 3×10 11 sequencing reads.
13 . The method of claim 7 , wherein the solid support comprises a plurality of oligonucleotides immobilized to a polymer.
14 . The method of claim 7 , wherein the solid support comprises a plurality of particles.
15 . The method of claim 7 , wherein the solid support comprises a plurality of oligonucleotides at a density of about 100 oligonucleotides per μm 2 to about 1,000,000 oligonucleotides per μm 2 .
16 . The method of claim 7 , wherein each template polynucleotide is not reseeded, reamplified, or both reseeded and reamplified after generating each sequencing read.
17 . The method of claim 9 , wherein the method generates a total number of sequencing reads greater than the number of optically resolvable features on the solid support.
18 . The method of claim 7 , wherein the about 20 to about 100 modified nucleotides are incorporated into the first sequencing primer, and about 20 to about 100 modified nucleotides are incorporated into the second sequencing primer, and about 20 to about 100 modified nucleotides are incorporated into the third sequencing primer.
19 . The method of claim 7 , wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide each comprise an amplification primer binding sequence, wherein said amplification primer binding sequence is the same.
20 . The method of claim 11 , wherein the optically resolvable features overlap by at least 25%.
21 . The method of claim 7 , comprising monitoring the sequential incorporation of 15 or more modified nucleotides prior to contacting the second polynucleotide with the second sequencing solution.
22 . The method of claim 7 , comprising monitoring the sequential incorporation of 15 to 100 modified nucleotides prior to contacting the second polynucleotide with the second sequencing solution.
23 . A method of sequencing three or more populations of polynucleotides on a solid support, wherein each population comprises a different sequencing primer binding sequence and a different template sequence, said method comprising:
(a) simultaneously amplifying three or more populations of polynucleotides on the solid support comprising a plurality of amplification primers attached to the solid support; and
(b) sequentially generating sequencing reads for each population of polynucleotides, wherein sequentially generating the sequencing reads comprises
i) hybridizing a sequencing primer to the sequencing primer binding sequence of a first population of polynucleotides;
ii) monitoring the sequential incorporation of 10 or more modified nucleotides, thereby generating a sequencing read; and
iii) after generating the sequencing read, repeating i) and ii) for each additional population of polynucleotides, wherein a different sequencing primer is hybridized to each sequencing primer binding sequence.
24 . The method of claim 23 , wherein monitoring the sequential incorporation of complementary nucleotides comprises a sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-binding process.
25 . The method of claim 23 , wherein monitoring the sequential incorporation of one or more modified nucleotides comprises
incorporating 10 or more modified nucleotides into a first sequencing primer with a polymerase to create a first extension strand, and detecting the one or more incorporated modified nucleotides in a first optically resolvable feature;
incorporating 10 or more modified nucleotides into a second sequencing primer with a polymerase to create a second extension strand, and detecting the one or more incorporated modified nucleotides in a second optically resolvable feature; and
incorporating 10 or more modified nucleotides into a third sequencing primer with a polymerase to create a third extension strand, and detecting the one or more incorporated modified nucleotides in a third optically resolvable feature; wherein the first, second, and third optically resolvable features overlap.
26 . The method of claim 25 , wherein the optically resolvable features overlap by at least 25%.
27 . The method of claim 1 , wherein
the first polynucleotide comprises a first sequencing primer binding sequence and a first template sequence,
the second polynucleotide comprises a second sequencing primer binding sequence and a second template sequence
the third polynucleotide comprises a third sequencing primer binding sequence and a third template sequence
wherein the first sequencing primer, second sequencing primer, and third sequencing primer each comprise different sequences, and the first template sequence, second template sequence, and third template sequence are distinct and comprise different sequences.
28 . The method of claim 3 , prior to generating the second sequencing read, cleaving and removing the first extension strand.
29 . The method of claim 3 , prior to generating the third sequencing read, cleaving and removing the second extension strand.
30 . The method of claim 23 , wherein the modified nucleotides each comprise a reversible terminator capable of being cleaved by the same cleaving agent.