METHOD FOR GENERATING A RNA-SEQUENCING LIBRARY

• US 20180230527A1
• Filed: 08/24/2016
• Published: 08/16/2018
• Est. Priority Date: 08/24/2015
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: FANG, Nan, NOLL, Bernhard, HEITZ, Katja
• Granted Patent: US 11,248,262 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/131 Terminal transferase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The invention refers to a novel method of preparing strand-specific RNA-sequencing libraries that can be used to identify DNA coding and non-coding strands that are transcribed mRNA Strand to RNA. Such strand-specific RNA-sequencing libraries are especially useful in discovering anti-sense RNA and non-coding RNA. Random primer oligonucleotides, covalently coupled to a moiety, which blocks ligation, are used for RT reaction or the subsequent generation of the second DNA strand so that only one strand of the generated double-stranded DNA is ligated to sequencing adapters at the 5′ nucleotide and sequenced by paired-end sequencing.

Claims

1. A method of RNA sequencing, comprising:

(i) providing RNA;

(ii) generating a single-stranded first DNA strand, which is complementary to the RNA of (i), by subjecting the RNA of (i) to reverse transcription by using a reverse transcriptase and a first set of oligonucleotide primers;

(iii) generating a double-stranded second DNA strand using a DNA polymerase, a second set of oligonucleotide primers, and the single-stranded first DNA generated in (ii);

(iv) ligating adapters to the double-stranded second DNA of (iii) to obtain a generated DNA; and

(v) sequencing the generated DNA;

wherein;

a) the first set of oligonucleotide primers comprises a moiety at its/their 5′

terminal nucleotide, which blocks ligation at a 5′

terminus of the single-stranded first DNA strand generated in (ii);

or

b) the second set of oligonucleotide primers comprises a moiety at its/their 5′

terminal nucleotide, which blocks ligation at a 5′

terminus of the double-stranded second DNA strand generated in (iii).

2. The method of claim 1, further comprising, prior to step (iv):

(iii)(a) end-repairing the double-stranded second DNA strand using a polynucleotide kinase and an enzyme with polymerase and exonuclease activities to obtain end-repaired DNA strands.

3. The method of claim 2, further comprising, after (iii)(a):

(iii)(b) adding a terminal adenine to a 3′

termini of the double-stranded second DNA by strand using a deoxynucleotidyl transferase enzyme, wherein the adapters comprise 3′

terminal thymines, which in (iv) ligate to double-stranded second DNA strand comprising 3′

terminal adenines.

5. The method of according to claim 1, wherein the oligonucleotide primers comprising the moiety covalently coupled to the 5′

terminal nucleotide are random oligonucleotide primers.

7. The method according to claim 1, wherein:

(i) the first set or the second set of oligonucleotide primers comprises at the 5′

terminal nucleotide a 5′

phosphate that is not free, wherein optionally a 5′

OH group or a 5′

phosphate group at the 5′

terminal nucleotide is covalently coupled to the moiety, which blocks ligation;

(ii) a base of the 5′

terminal nucleotide if the first set or the second set of oligonucleotide primers is not any of thymine, adenine, cytosine, guanine, and uracil;

(iii) one or both 2′

hydrogen(s) of a deoxyribose of the 5′

terminal nucleotide of the first set or the second set of oligonucleotide primers is/are replaced by another atom or a blocking moiety; and

/or

(iv) the first set or the second set of oligonucleotide primers comprises a 5′

terminal nucleotide having a pentose in a sterical conformation, which is other than that of ribose or deoxyribose in RNA or DNA.

8. The method according to claim 7, wherein the 5′

phosphate group of the deoxyribose of the 5′

terminal nucleotide of the second set of oligonucleotide primers, which blocks ligation, is esterified, or wherein the 5′

OH group of the 5′

terminal nucleotide of the second set of oligonucleotide primers, which blocks ligation, primer is esterified or etherified.

9. The method according to claim 7, wherein the 5′

phosphate group of the second set of oligonucleotide primers, which blocks ligation, is esterified by an alkyl or aryl alcohol, or wherein the 5′

OH group of the second set of oligonucleotide primers, which blocks ligation, is esterified by a monoalkyl phosphate, dialkyl phosphate, monoalkyl- or dialkyl phosphothionate, or by a boronic acid.

10. The method according to claim 9, wherein the alkyl or aryl alcohol comprises at least one additional functional group, which is selected from a mono- or polyether, mono- or polyester, carboxylate, primary amine or hydroxyl groups, or wherein the monoalkyl phosphate, dialkyl phosphate, monoalkyl or dialkyl phosphothionate, or the boronic acid comprises at least one additional functional group, which is selected from mono- or polyether, mono- or polyester, carboxylate, primary amine or hydroxyl groups.

11. The method according to claim 7, wherein the 5′

OH group of the deoxyribose of the 5′

terminal nucleotide of the second set of oligonucleotide primers, which blocks ligation, is esterified by a molecule selected from a group consisting of 5′

Spacer 18, 5′

Spacer 9, 5′

C3-Spacer, 5′

C6-Spacer, 5′

abasic residues, 5′

-5′

inverted nucleotides, and DADE-linker, 5′

C6-amino-linker, 5′

C12-amino-linker, and 5′

-biotinylated 5′

C6 and 5′

C12-amino-linker.

12. The method according to claim 7, wherein an unmodified 5′

terminal nucleotide is covalently coupled to any one of fludarabine, azathioprine, mercaptopurine, pentostatin, cladribine, floxuridine, gemcitabine, cytarabine, gemcitabine, capecitabine, and tegafur.

13. The method according to claim 1, wherein the generated single-stranded first DNA strand, which comprises the covalently coupled moiety, which blocks ligation at the 5′

terminus, further comprises a modification at its 3′

terminus, which blocks ligation at said 3′

terminus and which is introduced after the generation of the first single-stranded DNA strand.

4. A kit comprising:

(i) oligonucleotide primers comprising a moiety covalently coupled to a 5′

terminal nucleotide, which blocks ligation of DNA to sequencing adapters;

(ii) unmodified oligonucleotide primers;

(iii) a reverse transcriptase;

(iv) optionally a DNA polymerase;

(v) a polynucleotide kinase and an enzyme with polymerase and exonuclease activities;

(vi) optionally a deoxynucleotidyl transferase enzyme;

(vii) two adapters, which optionally comprise a terminal thymine, each of which is complementary to a surface-bound amplification primer, respectively; and

(viii) a ligase.

6. The method according to claim 4, wherein the reverse transcriptase is selected from a group consisting of retroviral reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase, murine leukemia virus reverse transcriptase, avian myeoloblastosis virus (AMV), bacterial reverse transcriptase, Tth DNA polymerase and a Taq DNA polymerase.