OLIGONUCLEOTIDE LIGATION, BARCODING AND METHODS AND COMPOSITIONS FOR IMPROVING DATA QUALITY AND THROUGHPUT USING MASSIVELY PARALLEL SEQUENCING

• US 20120028814A1
• Filed: 05/06/2011
• Published: 02/02/2012
• Est. Priority Date: 05/06/2010
• Status: Active Grant

• Current Assignee: Bioo Scientific Corporation (Revvity, Inc.)
• Original Assignee: Bioo Scientific Corporation (Revvity, Inc.)
• Inventors: Goldrick, Marianna, Toloue, Masoud M.
• Granted Patent: US 9,255,291 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

Described herein is a buffer concentration for highly efficient ligation of two oligonucleotides. The embodiments herein have led to the development of an optimized ligation step used in the sample preparation for sequencing reactions. Further, embodiments herein describe a high-throughput method for sequencing using barcodes or the purpose of multiplexing several samples simultaneously and novel methods for making targeted DNA libraries for re-sequencing on massively parallel next-generation sequencing platforms and for alternatives to gel-purification for recovering the desired templates from small RNA libraries for next generation sequencing.

Claims

1. A method of preparing nucleic acid molecules for sequencing reactions comprising:

ligating the nucleic acid molecules with a first oligonucleotide that is adenylated at the 3′

end in the absence of ATP, the first oligonucleotide having a known sequence; and

ligating the nucleic acid molecules with a second oligonucleotide that couples to the 5′

end of the nucleic acid molecules, the second oligonucleotide having a known sequence.

2. The method of claim 1, wherein the first oligonucleotide comprises a blocking group coupled to the 3′

end.

3. The method of claim 1, wherein ligating the nucleic acid molecules with the first oligonucleotide is performed in the presence of an RNA ligase capable of coupling the first oligonucleotide to the nucleic acid molecule in the absence of ATP.

4. The method of claim 3, wherein ligating the nucleic acid molecules with the first oligonucleotide is performed in the presence of a buffer comprising MgCl₂ and dithiothreitol (DTT).

5. The method of claim 4, wherein the concentration of MgCl₂ in the buffer ranges from about 1 mM to about 50 mM.

6. The method of claim 4, wherein the concentration of DTT in the buffer ranges from about 1 mM to about 15 mM.

7. The method of claim 1, wherein the first oligonucleotide and/or the second oligonucleotide comprise a barcode sequence portion and a portion having an oligonucleotide sequence complimentary to a reverse transcriptase primer.

8. The method of claim 7, wherein the barcode sequence portion comprises 2 to 12 nucleotides.

9. The method of claim 7, further comprising annealing the complementary reverse transcriptase primer to the 3′

end of the nucleic acid molecule after the first oligonucleotide is ligated to the nucleic acid molecule.

10. The method of claim 1, wherein the first oligonucleotide and the second oligonucleotide each comprise a barcode sequence portion and a portion having an oligonucleotide sequence complimentary to a reverse transcriptase primer, and wherein the barcode sequence portion of the first oligonucleotide is different from the barcode sequence portion of the second oligonucleotide.

11. The method of claim 1, further comprising annealing one or more oligonucleotides complementary to the first oligonucleotide-nucleic acid molecule junction and/or the second oligonucleotide-nucleic acid molecule junction.

12. The method of claim 1, wherein ligating the nucleic acid molecules with the first oligonucleotide is performed in the presence of polyethylene glycol (PEG).

13. The method of claim 1, wherein the nucleic acid molecules are small RNA molecules.

14. The method of claim 1, further comprising purifying the nucleic acid molecules ligated with the first and second oligonucleotides.

15. The method of claim 14, wherein purification of the ligated nucleic acid molecules comprises:

annealing a reaction mixture obtained after ligating the first and second oligonucleotides to the nucleic acid molecules with a third oligomer complementary for a nucleotide sequence formed by linkage of the first oligonucleotide to the second oligonucleotide; and

reacting the annealed mixture with a restriction endonuclease that is specific for a recognition site comprising the nucleotide sequence formed by linkage of the first oligonucleotide to the second oligonucleotide.

16. The method of claim 14, wherein purification of the ligated nucleic acid molecules comprises:

mixing the ligated nucleic acid molecules with one or more beads comprising an oligonucleotide sequence complementary to at least a portion of the first oligonucleotide sequence and/or at least a portion of the second oligonucleotide sequence;

separating the beads from the mixture;

removing the nucleic acid molecules from the beads.

17. A method of sequencing small RNA molecules comprising:

ligating the small RNA molecules with a first oligonucleotide that is adenylated at the 3′

end in the absence of ATP, the first oligonucleotide having a known sequence;

ligating the RNA molecules with a second oligonucleotide that couples to the 5′

end of the nucleic acid molecules, the second oligonucleotide having a known sequence;

reverse transcribing the ligated RNA molecule using a primer complementary to all or part of the ligated first oligonucleotide sequence and/or the ligated second oligonucleotide sequence;

amplifying the transcribed RNA to produce a mixture comprising multiple copies of the RNA molecule; and

determining the sequence of the RNA molecules in the mixture.