Multiplex barcoded Paired-End ditag (mbPED) library construction for ultra high throughput sequencing

1. An isolated barcoded Paired-End Ditag (bPED) nucleic acid fragment, comprising:

(a) a barcoded adaptor, comprising:

(i) a barcode, comprising at least one base pair of a known nucleotide sequence;

(ii) a 5′-flanking sequence, comprising a first restriction enzyme (RE) recognition site; and

(iii) a 3-flanking sequence, comprising a second RE recognition site and covalently linked to the 5′-flanking sequence via the barcode;

wherein the length of the barcoded adaptor is 18 or fewer base pairs, and the first and the second REs are the same or different and they each allow binding of specific restriction enzymes that lead to cleavage of a nucleic acid sequence at a defined distance from their respective recognition sites;

(b) a first tag; and

(c) a second tag, covalently linked to the first tag via the barcoded adaptor;

wherein the first tag corresponds to the 5′-end of a nucleic acid molecule, and the second tag corresponds to the 3′-end of the nucleic acid molecule, and the sequence of the nucleic acid molecule is unknown.

2. The isolated bPED nucleic acid fragment of claim 1 , wherein the lengths of the first and the second tags are no shorter than 14 bp.

3. The isolated bPED nucleic acid fragment of claim 1 , wherein the barcoded adaptor consists of:

(a) the barcode;

(b) the 5 flanking sequence, consisting of the first RE recognition site; and

(c) the 3-flanking sequence, consisting of the second RE recognition site.

4. The isolated bPED nucleic acid fragment of claim 1 , wherein the first and the second restriction enzyme recognition sites allow binding of specific restriction enzymes that lead to cleavage of a nucleic acid at ≧14 away from their respective recognition sites.

5. The isolated bPED nucleic acid fragment of claim 1 , wherein the length of the barcode is no shorter than 3 bp.

6. The isolated bPED nucleic acid fragment of claim 1 , wherein the first RE and/or the second RE are selected from the group consisting of MmeI, BpuEI, AcuI, and BsgI.

7. The isolated bPED nucleic acid fragment of claim 1 , wherein the barcoded adaptor comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-14.

8. The isolated bPED nucleic acid fragment of claim 1 , wherein the barcoded adaptor consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-14.

9. A method of generating the isolated bPED nucleic acid fragment of claim 1 , comprising the steps of:

(a) labeling a nucleic acid molecule of unknown sequence with a barcode by ligating the two ends of the nucleic acid molecule to a barcoded adaptor to obtain a circularized barcoded nucleic acid molecule, wherein the barcoded adaptor comprises:

(i) a barcode, comprising at least one base pair of a known nucleotide sequence;

(ii) a 5′-flanking sequence, comprising a first RE recognition site; and

(iii) a 3′-flanking sequence, comprising a second RE recognition site and covalently linked to the 5% flanking sequence via the barcode;

wherein the length of the barcode adaptor is 18 or fewer base pairs, and the first and the second REs are the same or different and they allow binding of specific restriction enzymes that lead to cleavage of a nucleic acid at a defined distance from their respective recognition sites; and

(b) digesting the circularized barcoded nucleic acid molecule with the first and second REs and lead to cleavage of the circularized barcoded nucleic acid molecule, and thereby generating the bPED nucleic acid fragment.

10. An isolated barcoded Paired-End Ditag (bPED) nucleic acid fragment, consisting of:

(a) a barcoded adaptor, consisting of:

(i) a barcode, consisting of at least one base pair of a known nucleotide sequence;

(ii) a 5′-flanking sequence, consisting of a first restriction enzyme (RE) recognition site; and

(iii) a 3′-flanking sequence, consisting of a second restriction enzyme (RE) site and covalently linked to the 5-flanking sequence via the barcode;

wherein the length of the barcode adaptor is 18 or fewer base pairs, and the first and the second REs are the same or different and they each allow binding of specific restriction enzymes that lead to cleavage of a nucleic acid sequence at a defined distance from their respective recognition sites;

(b) a first tag; and

(c) a second tag, covalently linked to the first tag via the barcoded adaptor;

wherein the first tag corresponds to the 5′-end of a nucleic acid molecule, and the second tag corresponds to the 3′-end of the nucleic acid molecule, and the sequence of the nucleic acid molecule is unknown.

11. The isolated bPED nucleic acid fragment of claim 1 , wherein the nucleic acid molecule is selected from the group consisting of cDNA, chromatin immunoprecipitation fragments, and genomic nucleic acid fragments.

12. The isolated bPED nucleic acid fragment of claim 10 , wherein the first and/or the second REs each lead to cleavage of a nucleic acid at ≧14 bp away from their respective recognition sites.

13. The isolated bPED nucleic acid fragment of claim 1 , wherein the nucleic acid molecule originates from a cancer cell, a normal cell or a single cell.

14. The isolated bPED nucleic acid fragment of claim 1 , wherein the first and the second REs are the same.

15. The isolated bPED nucleic acid fragment of claim 1 , wherein the nucleic acid molecule originates from a cancer cell.

16. The isolated bPED nucleic acid fragment of claim 10 , wherein the nucleic acid molecule is selected from the group consisting of cDNA, chromatin immunoprecipitation fragments, and genomic nucleic acid fragments.

17. The isolated bPED nucleic acid fragment of claim 10 , wherein the nucleic acid molecule originates from a cancer cell, a normal cell or a single cell.