Sequence tags
Provided herein is a new class of nucleic acid tagging molecules which are essentially free of homopolymer stretches. The tagging molecules can tag a plurality of individual target molecules for detection with a high degree of accuracy. The tagging molecules can be used to tag at least 105 or 106 individual target molecules. The tagged individual target molecules can be subjected to high throughput sequence analysis.
1. A method for determining the sequences of a plurality of individual target nucleic acid molecules comprising the steps of:
a. tagging each of said plurality of individual nucleic acid target molecules with an individual representative of a plurality of tagging oligonucleotides to generate a plurality of tagged individual target nucleic acid molecules, said plurality of tagging oligonucleotides:
i. comprising at least 10 5 individual representatives, each individual representative comprising a degenerated sequence segment,
wherein the degenerated sequence segment comprises one or more identical subsegments having a sequence X1-X2-X3-X4,
wherein X1 is a degenerated base region comprising a first set of three different bases or base analogs, X2 is a degenerated base region comprising a second set of three different bases or base analogs, X3 is a degenerated base region comprising a third set of three different bases or base analogs, and X4 is a degenerated base region comprising a fourth set of three different bases or base analogs, wherein said first, second, third and fourth sets are different from each other; and
ii. not comprising an individual representative with a homopolymer stretch longer than 3 nucleotides in the degenerated sequence segment;
b. amplifying said plurality of tagged individual target nucleic acid molecules, thereby generating amplified tagged individual target nucleic acid molecules; and
c. sequencing said amplified tagged individual target nucleic acid molecules, thereby determining the sequences of the plurality of target nucleic acid molecules.
2. The method according to claim 1 , wherein said plurality of individual target nucleic acid molecules comprise one or more identical nucleic acid analyte regions.
3. The method according to claim 2 , further comprising a step of generating a consensus sequence for each of the one or more identical nucleic acid analyte regions from the sequences of the plurality of individual target nucleic acid molecules.
4. The method of claim 1 , wherein the degenerated sequence segment comprises at least 2 subsegments.
5. The method of claim 4 , wherein the degenerated sequence segment comprises at least 2 identical subsegments.
6. The method of claim 1 , wherein the tagging is executed by ligating an individual representative of the plurality of tagging oligonucleotides to each of the plurality of individual target molecules.
7. The method of claim 1 , wherein the tagging is executed by hybridizing a target binding site located downstream of the degenerated sequence segment of each individual representative of the plurality of tagging oligonucleotides to each of the plurality of individual target molecules.
8. The method of claim 7 , further comprising elongating said plurality of tagging oligonucleotides by performing a target dependent DNA polymerase catalyzed primer extension reaction.