Sample preparation
The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.
1. A method of enriching, comprising:
a) providing i) an aqueous mix comprising PCR reagents, ii) template, iii) first and second PCR primers, said first PCR primers immobilized on emulsion beads for emulsion PCR, said second PCR primers comprising biotin, iv) an oil/detergent mix, and v) enrichment beads, wherein said enrichment beads comprise streptavidin;
b) adding said template, said first and second primers, said aqueous mix and said oil/detergent mix together so as to create one or more microemulsions, said one or more microemulsions comprising an aqueous compartment, each compartment comprising on average less than one template molecule;
c) temperature cycling said one or more microemulsions so as to amplify at least some of said template on at least some of said emulsion beads so as to create amplified product comprising biotin; and
d) enriching for beads with amplified template among said emulsion beads by contacting with said enrichment beads, wherein said emulsion beads with amplified template bind to said enrichment beads.
2. The method of claim 1 , wherein said enrichment beads comprise streptavidin and said binding to said enrichment beads in step d) proceeds through biotin-streptavidin binding.
3. The method of claim 1 , wherein said enrichment beads are magnetic.
4. The method of claim 1 , further comprising breaking said one or more microemulsions after step c) and before step d).
5. The method of claim 1 , wherein said template comprises sheared DNA fragments.
6. The method of claim 5 , wherein said sheared DNA fragments are attached to a linker with two primer recognition sequences.
7. The method of claim 6 , wherein said first primers are complementary to one of said primer recognition sequences and said second primers are complementary to the other primer recognition sequence.
8. A method of enriching, comprising:
a) providing i) an aqueous mix comprising PCR reagents, ii) template, iii) first and second PCR primers, said first PCR primers immobilized on emulsion beads for emulsion PCR, said second PCR primers comprising biotin, iv) an oil/detergent mix, and v) enrichment beads, wherein said enrichment beads comprise streptavidin;
b) adding said template, said first and second primers, said aqueous mix and said oil/detergent mix together so as to create one or more microemulsions, said one or more microemulsions comprising an aqueous compartment, each compartment comprising on average less than one template molecule;
c) temperature cycling said one or more microemulsions so as to amplify at least some of said template on at least some of said emulsion beads so as to create amplified product comprising biotin;
d) breaking said one or more microemulsions; and
e) enriching for beads with amplified template among said emulsion beads by contacting with said enrichment beads, wherein said emulsion beads with amplified template bind to said enrichment beads.
9. The method of claim 8 , wherein said enrichment beads comprise streptavidin and said binding to said enrichment beads in step d) proceeds through biotin-streptavidin binding.
10. The method of claim 8 , wherein said enrichment beads are magnetic.
11. The method of claim 8 , wherein said template comprises sheared DNA fragments.
12. The method of claim 11 , wherein said sheared DNA fragments are attached to a linker with two primer recognition sequences.
13. The method of claim 12 , wherein said first primers are complementary to one of said primer recognition sequences and said second primers are complementary to the other primer recognition sequence.