High density sequencing and multiplexed priming

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.

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.