Compositions and Methods for Constructing cDNA Libraries that Allow for Mapping the 5' and 3' Ends of RNAs

• US 20150099671A1
• Filed: 09/22/2014
• Published: 04/09/2015
• Est. Priority Date: 09/20/2013
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Moore, Melissa J., Heyer, Erin E., Ricci, Emiliano P., Ozadam, Hakan, Cenik, Can, Li, Xin
• Granted Patent: US 9,828,600 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

This disclosure provides methods and compositions for preparing and constructing cDNA libraries.

Claims

1. A method of optimizing the preparation of RNA molecules from a biological sample for sequencing, comprising of the steps of:

providing a biological sample that comprises RNA molecules;

ligating a DNA adaptor to the 3′

end of the RNA molecules, wherein the ligating is performed under one or more conditions that optimize the ligation reaction;

reverse transcribing the RNA molecules using a unique DNA primer under one or more conditions that optimize the reverse transcription reaction to produce single-stranded cDNA molecules;

purifying the cDNA molecules under one or more conditions that limit loss of the single-stranded cDNA molecules;

circularizing the purified cDNA molecules under one or more conditions that optimize the circularization reaction;

amplifying the circularized cDNA molecules under one or more conditions that optimize the amplification reaction,

thereby preparing RNA molecules from a biological sample for sequencing.

2. The method of claim 1, wherein the sequencing is deep sequencing.

3. The method of claim 1, wherein the first DNA adaptor is pre-adenylated.

4. The method of claim 1, wherein the purifying step is a gel purifying.

5. The method of claim 1, wherein the one or more conditions that optimize the ligation reaction is selected from the group consisting of:

carrying out the reaction in the presence of about 400 nM to about 700 nM of the first DNA adaptor;

carrying out the reaction at about 25 C to about 30 C for about 4 to about 6 hours.

6. The method of claim 1, wherein the one or more conditions that optimize the ligation reaction comprise carrying out the reaction in the presence of about 470 nM of the first DNA adaptor and incubating the reaction at about 30°

C. for about 6 hours.

7. The method of claim 1, wherein the one or more conditions that optimize the reverse transcription reaction are selected from the group consisting of:

carrying out the reaction in the presence of a three-fold dilution of the ligation reaction;

carrying out the reaction in the presence of about 333 nM of the unique DNA primer;

carrying out the reaction in the presence of an amount of unique DNA primer that is less than about 1;

1 relative to the amount of DNA adaptor used in the ligating step;

carrying out the reaction in the presence of about 3 units to about 6 units of a reverse transcriptase enzyme;

carrying out the reaction in the presence of about 5.33 units of a reverse transcriptase enzyme;

carrying out the reaction for about 30 min to about 1 hour at about 50 C to about 60 C;

carrying out the reaction for about 30 mins at about 55 C;

carrying out the reaction in the absence of any additional MgCl₂.

8. The method of claim 1, wherein the reverse transcriptase enzyme is SuperScript III.

9. The method of claim 1, wherein the one or more conditions that optimize the circularization reaction are selected from the group consisting of:

carrying out the reaction in the presence of all or essentially all of the RNA molecules obtained after the purifying step;

carrying out the reaction in the presence of about 1M betaine;

carrying out the reaction at about 60 C for about 2 to about 4 hours.

10. The method of claim 1, wherein the one or more conditions that optimize the circularization reaction comprise carrying out the reaction in the presence of all or essentially all of the RNA molecules obtained after the purifying step in the reaction, in the presence of about 1M betaine, at about 60°

C. for about 3 hours.

11. The method of claim 1, wherein the conditions that optimize the amplification reaction comprise carrying out the reaction in the presence of the circularization reaction at about 20% of the total reaction volume.

12. A method of optimizing the preparation of RNA molecules from a biological sample for sequencing, consisting essentially of the steps of:

providing a biological sample comprising RNA molecules;

ligating a first DNA adaptor to the 3′

end of the RNA molecules, wherein the ligating is performed under conditions that optimize the ligation reaction, wherein the conditions that optimize the ligation reaction comprise carrying out the reaction in the presence of about 470 nM of the first DNA adaptor and incubating the reaction at about 30 C for about 6 hours;

reverse transcribing the RNA molecules using a primer under conditions that optimize the reverse transcription reaction to produce single-stranded cDNA molecules, wherein the conditions that optimize the reverse transcription reaction comprise using about 5 units of SuperScript III reverse transcriptase and carrying out the reaction for about 30 mins at about 55 C in the absence of any additional MgCl₂, wherein the primer comprises a first portion that is complementary to the first DNA adaptor and a second portion that comprises a forward primer sequence joined to a reverse primer sequence by a flexible linker;

gel purifying the cDNA molecules;

circularizing the purified cDNA molecules under conditions that optimize the circularization reaction, wherein the conditions that optimize the circularization reaction comprise carrying out the reaction in the presence of all or essentially all of the RNA molecules obtained after the purifying step in the reaction, in the presence of about 1M betaine, at about 60 C for about 3 hours;

amplifying the circularized cDNA molecules under conditions that optimize the amplification reaction, wherein the conditions that optimize the amplification reaction comprise carrying out the reaction in the presence of the circularization reaction at about 20% of the total reaction volume,

thereby preparing RNA molecules from a biological sample for sequencing.

13. A method of preparing mRNA molecules in a biological sample for sequencing, comprising:

providing capped mRNA molecules from the biological sample;

ligating a first DNA adaptor to the 3′

ends of the capped mRNA molecules;

ligating a unique RNA adaptor to the 5′

ends of de-capped mRNA molecules;

fragmenting the mRNA molecules and ligating a second DNA adaptor to the newly-formed 3′

ends of the fragmented mRNA molecules;

reverse transcribing the fragmented mRNA molecules to produce single-stranded complementary DNA (cDNA) molecules;

circularizing the single-stranded cDNA molecules; and

amplifying the circularized cDNA molecules,

thereby preparing the mRNA molecules in the biological sample for sequencing.

14. The method of claim 13, wherein the sequencing captures both 5′

and 3′

ends of the mRNA molecules.

15. The method of claim 13, wherein the sequencing determines the length of the polyA tail of the mRNA molecules.

16. The method of claim 13, wherein the sequencing is deep sequencing.

17. The method of claim 13, wherein the sequencing is paired-end sequencing.

18. The method of claim 13, wherein the first DNA adaptor is pre-adenylated.

19. The method of claim 13, wherein the second DNA adaptor is pre-adenylated.

20. The method of claim 13, wherein the cap is removed from the mRNA molecules using tobacco acid pyrophosphatase (TAP).

21. The method of claim 13, wherein the mRNA molecules are fragmented using alkaline hydrolysis.

22. A method of preparing non-coding RNA molecules in a biological sample for sequencing, comprising:

providing non-capped non-coding RNA molecules from the biological sample;

ligating a first DNA adaptor to the 3′

ends of the non-coding RNA molecules;

ligating a unique RNA adaptor to the 5′

ends of the non-coding RNA molecules;

fragmenting the non-coding RNA molecules and ligating a second DNA adaptor to the newly-formed 3′

ends of the fragmented non-coding RNA molecules;

reverse transcribing the fragmented non-coding RNA molecules to produce single-stranded complementary DNA (cDNA) molecules;

circularizing the single-stranded cDNA molecules; and

amplifying the circularized cDNA molecules,

thereby preparing the non-coding RNA molecules in the biological sample for sequencing.