Primer extension target enrichment and improvements thereto including simultaneous enrichment of dna and rna

A method of enriching a target polynucleotide from a sample, the method comprising:

• a) providing a sample comprising the target polynucleotide in both single stranded RNA and double stranded DNA form;
• b) hybridizing a first target-specific primer to the single-stranded RNA form of the target polynucleotide wherein the primer comprises a target-binding region and a region of complementarity to an adaptor;
• c) extending the hybridized first target-specific primer with a reverse transcriptase to form a double-stranded RNA-DNA hybrid comprising an RNA strand and a first primer extension product;
• d) subjecting the sample to double stranded nucleic acid denaturation conditions;
• e) hybridizing a second target-specific primer to the denatured DNA wherein the primer comprises a target-binding region and a region of complementarity to the adaptor;
• f) extending the hybridized second target-specific primer with a DNA polymerase to form a second primer extension product;
• g) contacting the sample with the adaptor comprising a longer strand a shorter strand wherein the longer strand comprises a region complementary to the region in the target-specific primer, and the shorter strand comprises a universal priming site;
• h) hybridizing the adaptor to the first and second primer extension products;
• i) ligating one strand of the adaptor to the first and the second primer extension products to form ligation products;
• j) hybridizing the third target-specific primer to the first primer extension product and a fourth primer to the second primer extension product wherein the third and fourth target-specific primers comprise a target-binding site and a universal priming site;
• k) amplifying the ligation products utilizing primers hybridizing to the universal priming sites.

A method of sequencing a target polynucleotide from a sample, the method comprising:

• a) providing a sample comprising the target polynucleotide in both single stranded RNA and double stranded DNA form;
• b) hybridizing a first target-specific primer to the single-stranded RNA form of the target polynucleotide wherein the primer comprises a target-binding region and a region of complementarity with an adaptor;
• c) extending the hybridized first target-specific primer with a reverse transcriptase to form a double-stranded RNA-DNA hybrid comprising an RNA strand and a first primer extension product;
• d) subjecting the sample to double stranded nucleic acid denaturation conditions;
• e) hybridizing the second target-specific primer to the denatured DNA;
• f) extending the hybridized second target-specific primer, wherein the primer comprises a target-binding region and a region of complementarity with an adaptor with a DNA polymerase to form a second primer extension product;
• g) contacting the sample with the adaptor comprising a longer strand a shorter strand wherein the longer strand comprises a region complementary to the region in the first and second target-specific primers, and the shorter strand comprises a universal priming site;
• h) hybridizing the adaptor to the first and second primer extension products;
• i) ligating one strand of the adaptor to the first and the second primer extension products to form ligation products;
• j) hybridizing the third target-specific primer to the first primer extension product and the fourth target-specific primer to the second primer extension product wherein the third and fourth target-specific primers comprise a target-binding site and a universal priming site;
• k) amplifying the ligation products utilizing primers hybridizing to the universal priming sites;
• l) sequencing the amplified ligation products from step k).

A method of enriching a target polynucleotide from a sample, the method comprising:

• a) providing a sample comprising the target polynucleotide in both single stranded RNA and double stranded DNA form;
• b) hybridizing a first target-specific primer to the single-stranded RNA form of the target polynucleotide wherein the primer comprises a target-binding region;
• c) extending the hybridized first target-specific primer with a reverse transcriptase to form a double-stranded RNA-DNA hybrid comprising an RNA strand and a first primer extension product;
• d) subjecting the sample to double stranded nucleic acid denaturation conditions;
• e) hybridizing a second target-specific primer to the denatured DNA wherein the primer comprises a target-binding region and a region of complementarity to an adaptor;
• f) hybridizing a third target-specific primer to the first primer extension product wherein the primer comprises a target-binding region and a region of complementarity to the adaptor;
• g) extending the hybridized second and third target-specific primers with a DNA polymerase to form a second and third primer extension products;
• h) contacting the sample with the adaptor comprising a longer strand a shorter strand wherein the longer strand comprises a region complementary to the region in the target-specific primers, and the shorter strand comprises a universal priming site;
• i) hybridizing the adaptor to the second and third primer extension products;
• j) ligating one strand of the adaptor to the second and third primer extension products to form ligation products;
• k) hybridizing a fourth target-specific primer to the second primer extension product and a fifth primer to the third primer extension product wherein the fourth and fifth target-specific primers comprise a target-binding site and a universal priming site;
• and
• l) amplifying the ligation products utilizing primers hybridizing to the universal priming sites.