Molecular identity tags and uses thereof in identifying intermolecular ligation products

• US 10,385,334 B2
• Filed: 05/30/2014
• Issued: 08/20/2019
• Est. Priority Date: 05/31/2013
• Status: Active Grant

• Current Assignee: Si Lok
• Original Assignee: Si Lok
• Inventors: Lok, Si
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US14/894,653
• Publication Number:
  US 20160108394A1
• Time in Patent Office: 1,908 Days
• Field of Search: None
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Abstract

Molecular identity tags and methods of marking target nucleic acids with molecular identity tags for identifying intermolecular ligation products. Terminal regions of linear target nucleic acids are marked with pairs of distinguishable molecular identity tags. Each linear target nucleic acid is marked with only one distinguishable pair. The terminal ends of the marked target nucleic acids are joined to generate circularized nucleic acids, thereby juxtaposing two molecular identity tags across the joined terminal ends. Circularized nucleic acids or downstream products thereof comprising juxtaposed unpaired molecular identity tags constitute intermolecular nucleic acid products that can be identified and eliminated from subsequent analyses of the nucleic acids. The molecular identity tags may comprise physically linked and non-physically linked pairs. Physically linked molecular identity tags designed for production and analysis of mate-pair libraries with next-generation sequencing platforms are provided.

Claims

1. A method of identifying intermolecular ligation products, comprising:

marking linear target nucleic acids with multiple, mutually distinguishable pairs of molecular identity tags to yield marked target nucleic acids, wherein the marking comprises marking terminal regions of each linear target nucleic acid with one of the pairs of molecular identity tags, wherein a first terminal region of each target nucleic acid is marked with a first member of the one of the pairs and a second terminal region of each target nucleic acid is marked with a second member of the one of the pairs;

joining terminal ends of the marked target nucleic acids or products of the marked target nucleic acids to yield marked nucleic acid products, each marked nucleic acid product comprising two of the molecular identity tags juxtaposed across the joined terminal ends, wherein the marked nucleic acid products include one or more marked nucleic acid products comprising only juxtaposed paired molecular identity tags and one or more marked nucleic acid products comprising juxtaposed unpaired molecular identity tags, wherein any marked nucleic acid product comprising juxtaposed unpaired molecular identity tags constitutes an intermolecular nucleic acid product; and

parsing the one or more marked nucleic acid products comprising only juxtaposed paired molecular identity tags, or products thereof, from the one or more marked nucleic acid products comprising juxtaposed unpaired molecular identity tags, or products thereof.

2. The method of claim 1 wherein the parsing comprises sequencing the marked nucleic acid products or products thereof.

7. The method of claim 1 wherein the marking comprises marking the terminal regions of each linear target nucleic acid with non-physically linked pairs of molecular identity tags.

8. The method of claim 7 wherein the marking the terminal regions of each linear target nucleic acid with non-physically linked pairs of molecular identity tags comprises:

dividing the linear target nucleic acids into first aliquots;

terminally attaching a different partial molecular identity tag to the divided target nucleic acids in each first aliquot to generate partially marked target nucleic acids;

pooling the partially marked target nucleic acids;

dividing the pooled, partially marked target nucleic acids into second aliquots; and

terminally attaching a different partial molecular identity tag to the divided, partially marked target nucleic acids in each second aliquot to generate the marked target nucleic acids.

9. The method of claim 1 wherein the joining further includes inserting a nucleic acid comprising a junction code between the joined terminal ends of the marked target nucleic acids or products of the marked target nucleic acids.

10. The method of claim 1 wherein the marking comprises marking the linear target nucleic acids with a plurality of nucleic acids, each of the plurality of nucleic acids comprising:

at least a first nucleic acid strand, the first nucleic acid strand on each of the plurality of nucleic acids comprising;

a first molecular identity tag;

a first primer-binding site;

a second molecular identity tag; and

a reverse complement of a second primer-binding site;

wherein;

the first molecular identity tag is located on the first nucleic acid strand in a 5′

position with respect to the first primer-binding site;

the second molecular identity tag is located on the first nucleic acid strand in a 3′

position with respect to the reverse complement of the second primer-binding site; and

the first molecular identity tag and the first primer-binding site are located on the first nucleic acid strand in a 5′

position with respect to the second molecular identity tag and the reverse complement of the second primer-binding site;

wherein;

the first primer-binding sites in the plurality of nucleic acids are identical or substantially identical with each other;

the reverse complements of the second primer-binding sites in the plurality of nucleic acids are identical or substantially identical with each other;

the first molecular identity tags in a set of at least two of the plurality of nucleic acids are distinguishable from each other; and

the second molecular identity tags in the set of the plurality of nucleic acids are distinguishable from each other.

11. The method of claim 10 wherein the plurality of nucleic acids comprise multiple copies of each of the at least two of the plurality of nucleic acids in the set, wherein each copy is a nucleic acid with a sequence identical to one of the at least two of the plurality of nucleic acids in the set.

12. The method of claim 10 wherein each of the plurality of nucleic acids is a linear nucleic acid including a first terminus and a second terminus.

13. The method of claim 12, wherein the first molecular identity tag is located between the first terminus and the second molecular identity tag, and the second molecular identity tag is located between the second terminus and the first molecular identity tag.

14. The method of claim 13 wherein each of the first nucleic acid strands comprises from 0 to about 90 bases between a 5′

end of the first molecular identity tag and the first terminus, and further comprises from 0 to about 90 bases between the second terminus and a 3′

end of the second molecular identity tag.

15. The method of claim 13 wherein each of the first nucleic acid strands comprises from 2 to about 100 bases between a 5′

end of the first primer-binding site and the first terminus, and further comprises from 2 to about 100 bases between the second terminus and a 3′

end of the reverse complement of the second primer-binding site.

16. The method of claim 10 wherein each of the first nucleic acid strands comprises from about 30 bases to about 10 kilobases.

17. The method of claim 10 wherein the first molecular identity tags and the second molecular identity tags each independently have a length of from about 2 to about 30 bases.

18. The method of claim 10 wherein each of the plurality of nucleic acids includes a second nucleic acid strand that comprises a reverse complement of the first molecular identity tag, a reverse complement of the first primer-binding site, the second primer-binding site, and a reverse complement of the second molecular identity tag.

19. The method of claim 10 wherein the set of the nucleic acids includes at least 10 of the plurality of nucleic acids, wherein the first molecular identity tags in each of the at least 10 of the plurality of nucleic acids in the set are distinguishable from each other, and wherein the second molecular identity tags in each of the at least 10 of the plurality of nucleic acids in the set are distinguishable from each other.

3. A method of identifying intermolecular ligation products, comprising:

marking linear target nucleic acids with multiple, mutually distinguishable pairs of molecular identity tags to yield marked target nucleic acids, wherein the marking comprises marking terminal regions of each linear target nucleic acid with one of the pairs of molecular identity tags, wherein a first terminal region of each target nucleic acid is marked with a first member of the one of the pairs and a second terminal region of each target nucleic acid is marked with a second member of the one of the pairs;

joining terminal ends of the marked target nucleic acids or products of the marked target nucleic acids to yield circularized, marked nucleic acid products, each marked nucleic acid product comprising two of the molecular identity tags juxtaposed across the joined terminal ends, wherein any marked nucleic acid product comprising juxtaposed unpaired molecular identity tags constitutes an intermolecular nucleic acid product;

linearizing the circularized, marked nucleic acid products to yield one or more linearized, marked nucleic acids comprising only juxtaposed paired molecular identity tags and one or more linearized, marked nucleic acids comprising juxtaposed unpaired molecular identity tags; and

parsing the one or more linearized, marked nucleic acids comprising only juxtaposed paired molecular identity tags from the one or more linearized, marked nucleic acids comprising juxtaposed unpaired molecular identity tags.

4. The method of claim 3 wherein the linearizing is selected from the group consisting of fragmenting the circularized, marked nucleic acids and amplifying linear portions of the circularized, marked nucleic acids.

5. A method of identifying intermolecular ligation products, comprising:

marking linear target nucleic acids with multiple, mutually distinguishable pairs of molecular identity tags to yield marked target nucleic acids, wherein the marking comprises marking terminal regions of each linear target nucleic acid with one of the pairs of molecular identity tags, wherein a first terminal region of each target nucleic acid is marked with a first member of the one of the pairs and a second terminal region of each target nucleic acid is marked with a second member of the one of the pairs; and

joining terminal ends of the marked target nucleic acids or products of the marked target nucleic acids to yield marked nucleic acid products, each marked nucleic acid product comprising two of the molecular identity tags juxtaposed across the joined terminal ends, wherein any marked nucleic acid product comprising juxtaposed unpaired molecular identity tags constitutes an intermolecular nucleic acid product,

wherein;

each pair of molecular identity tags is physically linked;

the marking comprises marking the terminal regions of each linear target nucleic acid with one of the physically linked pairs of molecular identity tags to yield end-coupled, marked target nucleic acids, wherein the physically linked pairs of molecular identity tags are physically linked prior to and during the marking;

the method further comprises excising a target nucleic acid portion from each of the end-coupled, marked target nucleic acids to yield sequence-excised, marked target nucleic acids; and

the joining comprises circularizing the sequence-excised, marked target nucleic acids to yield the marked nucleic acid products.

6. The method of claim 5 wherein the marking comprises marking the linear target nucleic acids with a plurality of nucleic acids, each of the plurality of nucleic acids comprising:

at least a first nucleic acid strand, the first nucleic acid strand on each of the plurality of nucleic acids comprising;

a first molecular identity tag;

a first primer-binding site;

a second molecular identity tag; and

a reverse complement of a second primer-binding site;

wherein;

the first molecular identity tag is located on the first nucleic acid strand in a 5′

position with respect to the first primer-binding site;

the second molecular identity tag is located on the first nucleic acid strand in a 3′

position with respect to the reverse complement of the second primer-binding site; and

the first molecular identity tag and the first primer-binding site are located on the first nucleic acid strand in a 5′

position with respect to the second molecular identity tag and the reverse complement of the second primer-binding site;

wherein;

the first primer-binding sites in the plurality of nucleic acids are identical or substantially identical with each other;

the reverse complements of the second primer-binding sites in the plurality of nucleic acids are identical or substantially identical with each other;

the first molecular identity tags in a set of at least two of the plurality of nucleic acids are distinguishable from each other; and

the second molecular identity tags in the set of the plurality of nucleic acids are distinguishable from each other.