Nucleic acid analysis using sequence tokens
The present invention provides methods and compositions for tagging nucleic acid sequence fragments, e.g., a set of nucleic acid sequence fragments from a single genome, with one or more unique members of a collection of oligonucleotide tags, or sequence tokens, which, in turn, can be identified using a variety of readout platforms. As a general rule, a given sequence token is used once and only once in any tag sequence. In addition, the present invention also provides methods for using the sequence tokens to efficiently determine variations in nucleotide sequences in the associated nucleic acid sequence fragments.
1. A composition comprising a mixture of polynucleotides, wherein the polynucleotides in the mixture are made by combining a plurality of different polynucleotide samples, wherein:
each of the different samples comprises non-overlapping segments of a genome, wherein each of the non-overlapping segments is attached to a sequence tag that identifies the sample from which that segment is derived; and
each of the sequence tags comprises two or more unique token sequences, wherein the token sequences are chosen from different sets of token sequences, and within each set, the token sequences are different from one another and are designed to not cross hybridize with one another or their complements.
2. The composition of claim 1 , wherein said genome is a mammalian genome.
3. The composition of claim 1 , wherein said genome is a human genome.
4. The composition of claim 1 , wherein said genome is a viral genome.
5. The composition of claim 1 , wherein said plurality of different polynucleotide samples is at least 10 different polynucleotide samples.
6. The composition of claim 1 , wherein each of said sequence tags is at least 9 bases in length.
7. The composition of claim 1 , wherein each token sequence within each set has the same number of s bases and w bases, wherein s is G or C and w is A or T.
8. The composition of claim 1 , wherein each different token sequence has no adjacent GC pairs.
9. The composition of claim 1 , wherein no token sequence is contained in two different sets of token sequences.
10. The composition of claim 1 , wherein each of the different token sequences comprises a unique combination of triplet units selected from multiple different groups of triplet units, wherein each of said groups of triplet units has four members, and wherein each member of a group of triplet units is different from the other members of the same group by at least 2 out of 3 bases.
11. The composition of claim 10 , wherein each of the different token sequences comprises a unique combination of at least three of the triplet units.
12. The composition of claim 10 , wherein the multiple groups of triplet units are:
Group I (sww): CAA, GAT, CTT, GTA;
Group II (wsw): TGA, AGT, TCT, ACA;
Group III (wws): TAG, AAC, TTC, ATG;
Group IV (www): TAA, AAT, TTT, ATA;
and
Group V (sws): CAG, GAC, CTC, GTG.
13. The composition of claim 11 , wherein the polynucleotides from each of the plurality of polynucleotide samples comprises three or more token sequences attached thereto.
14. The composition of claim 1 , wherein the two or more token sequences are not adjacent to one another.
15. The composition of claim 1 , wherein the non-overlapping segments are restriction enzyme fragments.
16. The composition of claim 1 , wherein the non-overlapping segments are amplicons.