Multifunctional primers for paired sequencing reads

A method of obtaining paired sequencing reads of a target nucleic acid, comprising

• (a) contacting a single-stranded target nucleic acid flanked at its 5′ end by a 5′ adapter immobilized to a support and at its 3′ end by a 3′ adapter with a forward primer comprising a 3′ target binding region and a 5′ stem loop to form a reaction mix, wherein the target binding region binds the 3′ adapter and wherein the target binding region comprises a cleavable site;
• (b) ligating a free 5′ end of the stem loop to the 3′ adapter;
• (c) conducting a sequencing-by-extension reaction, wherein the target binding region primes synthesis of a first complementary strand to the target nucleic acid and provides a sequencing read of the target nucleic acid;
• cleaving the cleavable site, and conducting a further extension reaction wherein the remainder of the target binding region after cleavage primes synthesis of a further complementary strand to the target nucleic acid;
• (d) contacting the reaction mix with a loop primer that anneals to the loop region of the stem loop;
• (e) conducting an extension reaction wherein the loop primer primes synthesis of a second complementary strand to the target nucleic acid, which displaces the further complementary strand from duplexing with the target nucleic acid;
• (f) contacting the reaction mix with a reverse primer, which anneals to a complement of the 5′ adapter at the 3′ end of the displaced further complementary strand;
• and
• (g) conducting a sequencing-by-extension reaction, wherein the reverse primer primes synthesis of a strand complementary to the displaced further complementary strand and provides a sequencing read of the displaced further complementary strand.

A method of obtaining paired sequencing reads from a target nucleic acid, comprising

• (a) contacting a single-stranded target nucleic acid flanked at its 5′ end by a 5′ adapter immobilized to a support and at its 3′ end by a 3′ adapter with a forward primer comprising first and second 3′ target binding regions flanking an intervening region to form a reaction mix, wherein the first and second target binding regions bind complementary sites in the 3′ adapter molecule, the respective sites being distal and proximal to the 3′ end of the 3′ adapter;
• (b) conducting an extension reaction, wherein the first target binding region primes synthesis of a first complementary strand to the target nucleic acid and optionally provides a sequencing read of the target nucleic acid;
• (c) conducting a second extension reaction wherein the second target binding region primes synthesis of a second complementary strand to the target nucleic acid, which displaces the complementary strand from duplexing with the target nucleic acid;
• (d) contacting the reaction mix with a reverse primer, which anneals with a complement of the 5′ adapter at the 3′ end of the displaced first complementary strand;
• and
• (e) conducting a sequencing-by-extension reaction, wherein the reverse primer primes synthesis of a strand complementary to the displaced first complementary strand and provides a sequencing read of the displaced first complementary strand.