Methods for identifying low-abundance polynucleotides and related compositions

A method for preparing a selected polynucleotide pool from a polynucleotide sample comprising:

• a) synthesizing first antisense polynucleotide strands from sense polynucleotides of, or prepared from, the polynucleotide sample using an antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′ of the antisense primer;
• b) adding a universal primer site to the 3′ ends of the first antisense polynucleotide strands;
• c) diluting the first antisense polynucleotide strands to substantially eliminate at least some low-abundance first antisense polynucleotide strands;
• and
• d) producing first double-stranded polynucleotides from the remaining first antisense polynucleotide strands, wherein the first double-stranded polynucleotides are enriched in high-abundance polynucleotide sequences relative to the polynucleotide sample.

A method for preparing a selected polynucleotide pool from a polynucleotide sample comprising:

• a) hybridizing first antisense polynucleotide strands prepared from a first polynucleotide sample to sense polynucleotide strands of, or prepared from, a second polynucleotide sample, wherein the first antisense polynucleotide strands are enriched in high-abundance polynucleotide sequences relative to the first polynucleotide sample, thereby producing unhybridized sense polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the second polynucleotide sample;
• and
• b) synthesizing second antisense polynucleotide strands from the unhybridized sense polynucleotide strands using an antisense primer or an antisense primer complex, said antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′ of the antisense primer;
• c) adding a universal primer site to the 3′ ends of the second antisense polynucleotide strands;
• d) producing double-stranded polynucleotides from the second antisense polynucleotide strands.

A method for preparing a selected polynucleotide pool from a polynucleotide sample comprising:

• a) synthesizing first antisense polynucleotide strands from sense polynucleotides of, or prepared from, the polynucleotide sample;
• b) diluting the first antisense polynucleotide strands to substantially eliminate at least some low-abundance first antisense polynucleotide strands;
• and
• c) producing first double-stranded polynucleotides from the remaining first antisense polynucleotide strands, wherein the first double-stranded polynucleotides are enriched in high-abundance polynucleotide sequences relative to the polynucleotide sample;
• d) producing second antisense polynucleotide strands from the first double-stranded polynucleotides;
• e) contacting the second antisense polynucleotide strands with sense polynucleotide strands of, or prepared from, the polynucleotide sample under hybridization conditions to form a hybridization mixture, thereby producing unhybridized sense polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the polynucleotide sample;
• f) synthesizing third antisense polynucleotide strands from the unhybridized sense polynucleotide strands;
• g) producing second double-stranded polynucleotides from the third antisense polynucleotide strands.