Method and reagent for constructing nucleic acid double-linker single-strand cyclical library

A method for constructing a library of single-stranded cyclic nucleic acid fragments having double adaptors, comprising the following steps carried out in the following order:

• (1) disrupting a nucleic acid into nucleic acid fragments for library construction and selecting disrupted fragments using magnetic bead purification or gel recovery;
• (2) subjecting the fragments to dephosphorylation and end repairing reactions;
• (3) ligating a 5′ adaptor A sequence to the fragments, wherein the 5′ adaptor A sequence comprise a strand with 5′ end phosphorylation modification and the other strand with 3′ end dideoxy modification and uracil (U) base sites;
• (4) performing a one-step reaction of Uracil-Specific Excision Reagent enzyme digestion and phosphorylation using Uracil-Specific Excision Reagent (USER) enzyme and T4 polynucleotide kinase;
• (5) ligating a first 3′ L-type adaptor sequence to the 5′ end of each strand of the phosphorylated nucleic acid fragments;
• (6) performing a first polymerase chain reaction (PCR) amplification to obtain first products having the first adaptor sequence at both ends, wherein the primer sequences used in the first PCR have a U base site and have a nickase recognition sequence, and one of the primers has a biotin label;
• followed by purification using magnetic beads;
• (7) digesting the first products with a Uracil-Specific Excision Reagent (USER) enzyme to remove the U base site in the primer sequences to generate sticky ends and generate or do not generate a gap;
• (8) performing a double-stranded cyclization of the digested first products to generate cyclic nucleic acid molecules, which having a nickase recognition sequence on one strand and a gap on the other strand or having a nickase recognition sequence but not a gap on both strands, such that cyclization occurs due to the complementarity of the sticky ends to generate cyclized nucleic acid molecules;
• (9) performing linear digestion by treating the cyclic nucleic acid molecules having a nickase recognition sequence on one strand and a gap on the other strand or having a nickase recognition sequence but not a gap on both strands with a nickase to generate a nick;
• (10) performing magnetic purification by binding the treated cyclic nucleic acid molecules to streptavidin magnetic beads via the biotin label on the cyclic nucleic acid molecules;
• and allowing a solid phase carrier harboring a second affinity label to bind to the cyclic nucleic acid molecules;
• (11) enzymatically digesting the nickase recognition sequence with nickase to get a nick on each single-strand to provide an initiation site for controlled nick translation (CNT), wherein the enzymatic reaction proceeds on the magnetic beads without eluting the nucleic acids from the magnetic beads;
• (12) initiating a controlled nick translation reaction from the nick and/or gap by using the cyclic nucleic acid molecules bound to the streptavidin magnetic beads, wherein in the controlled gap translation reaction, the length of the gap translation fragments generated is controlled by controlling at least one factor selected from the group consisting of the molar ratio of deoxynucleotide triphosphates (dNTPs) to the nucleic acid molecules as template, the enzymatic reaction temperature, and the enzymatic reaction time, to generate nucleic acid fragments with a length in the range of 50-250 base pairs (bp);
• (13) digesting and removing the portion of the respective cyclic nucleic acid molecules that does not undergo the controlled gap translation reaction to obtain linear nucleic acid molecules;
• (14) ligating a second adaptor sequence bubble-type adaptor comprising a top strand and a bottom strand which are complementary to one another to both ends of the linear nucleic acid molecules, wherein the bottom strand having U base sites;
• (15) performing Uracil-Specific Excision Reagent (USER) enzyme digestion of the U base site;
• (16) performing a second polymerase chain reaction amplification to obtain second products having the second adaptor sequence at both ends;
• (17) denaturing the second products to obtain single-stranded nucleic acid molecules, and cyclizing one of the single-stranded nucleic acid molecules with a mediating sequence complementary to both ends of the single-stranded nucleic acid molecule to obtain the library of single-stranded cyclic nucleic acid fragments having double adaptors to produce nucleic acid fragments in a size range of 50 bp to 250 bp, and wherein the steps of the method provide fragments suitable for next-generation sequencing on a Complete Genomics sequencing platform.