Methods and kits for reducing adapter-dimer formation
The current teachings relate to methods for reducing adapter-dimer formation, particularly when preparing nucleic acids of interest for subsequent amplification and/or sequencing. Also described are kits for use in performing certain disclosed methods.
1. A method for reducing adapter-dimer formation comprising:
combining a sample comprising target small ribonucleic acids (RNA), at least one deoxyribonucleic acid (DNA) 3′ adapter, and at least one first RNA ligase to form a first reaction composition;
incubating the first reaction composition under conditions suitable for first ligation products to be generated, to form a second reaction composition comprising first ligation products and at least some un-ligated 3′ adapters;
combining at least one oligonucleotide comprising a reverse transcription priming site with the second reaction composition to form a third reaction composition;
incubating the third reaction composition under conditions suitable for at least some of the oligonucleotides to anneal with at least some of the first reaction products and at least some of the un-ligated 3′ adapters to form 3′ adapter-oligonucleotide duplexes comprising a single-stranded 5′ overhang portion;
combining at least one DNA-dependent DNA polymerase to the third reaction composition and incubating under conditions suitable for the polymerase to convert at least some of the 3′ adapter-oligonucleotide duplexes comprising single-stranded 5′ overhang portions to double-stranded blunt ended adapter-oligonucleotide duplexes by end-filling;
combining at least one second ligase and at least one 5′ adapter to the third reaction composition comprising double-stranded adapter-oligonucleotide duplexes and first ligation products and incubating under conditions suitable for forming at least some second ligation products;
combining at least one reverse transcriptase with the second ligation products to generate double-stranded second ligation products, thereby reducing adapter-dimer formation.
2. The method of claim 1 , wherein the first ligase comprises T4 RNA ligase 2 or truncated T4 RNA ligase 2; the DNA-dependent DNA polymerase comprises T4 DNA polymerase; and the second ligase comprises T4 RNA ligase 1 or Methanobacterium thermoautotrophicum RNA ligase.
3. The method of claim 1 , wherein the oligonucleotide is annealed to the 3′ adapter prior to, during, or after ligating the 3′ adapter to a target ribonucleic acid.
4. The method of claim 1 wherein the 3′ adapter comprises 1-25 randomized bases at the 5′ end.
5. The method of claim 1 wherein the 5′ adapter comprises 1-25 randomized bases at the 3′ end.
6. The method of claim 1 , further comprising amplifying at least some of the double-stranded second ligation products to generate amplification products.
7. The method of claim 6 , wherein the amplifying comprises Polymerase Chain Reaction (PCR).
8. The method of claim 6 , further comprising separating at least some of the amplification products by size.
9. A method for reducing, adapter-dimer formation comprising:
combining a sample comprising target small ribonucleic acids, at least one 3′ adapter annealed to an oligonucleotide comprising a reverse transcription primer binding site, and at least one first ligase to form a first reaction composition, wherein the 3′ adapter annealed with the oligonucleotide comprises a single-stranded 5′ overhang portion;
incubating the first reaction composition under conditions suitable for first ligation products to be generated, to form a second reaction composition comprising first ligation products and at least some un-ligated 3′ adapters annealed to oligonucleotides;
combining at least one DNA-dependent DNA polymerase with the second reaction composition and incubating under conditions suitable for the polymerase to convert at least some of the single-stranded 5′ overhang portions of the 3′ adapters annealed to the oligonucleotides to double-stranded blunt ended adapter-oligonucleotide duplexes lacking overhang portions by end-filling;
combining at least one second ligase and at least one 5′ adapter to the second reaction composition comprising double-stranded adapter-oligonucleotide duplexes and first ligation products and incubating under conditions suitable for forming at least some second ligation products,
combining at least one reverse transcriptase with the second ligation products to generate double-stranded second ligation products,
thereby reducing adapter-dimer formation.
10. The method of claim 9 , wherein the first ligase comprises T4 RNA ligase 2 or truncated T4 RNA ligase 2; the DNA-dependent DNA, polymerase comprises T4 DNA polymerase; and the second ligase comprises T4 RNA ligase 1 or Methanobacterium thermoautotrophicum RNA ligase.
11. The method of claim 9 wherein the 3′ adapter comprises 1-25 randomized bases at the 5′ end.
12. The method of claim 9 wherein the 5′ adapter comprises 1-25 randomized bases at the 3′ end.
13. method of claim 9 , further comprising amplifying ask least some of the double-stranded second ligation products to generate amplification products.
14. The method ref claim 13 , wherein the amplifying comprises Polymerase Chain Reaction (PCR).