Methods related to DNA sequencing
In some aspects, the invention relates to a method for producing a barcoded DNA library, comprising: optionally fragmenting DNA to produce fragmented DNA; combining the DNA/fragments with control nucleotide sequences to produce a mixture of nucleic acids, each control nucleotide sequence comprising a variable nucleotide sequence, thereby barcoding the DNA/fragments; amplifying the mixture of nucleic acids to produce amplified nucleic acids; isolating a plurality of nucleic acids from the amplified nucleic acids; and rearranging the nucleic acids of the plurality, thereby producing the barcoded DNA library.
1. A method for producing a barcoded DNA library for obtaining a consensus sequence from multiple short reads by using molecular recombination to bring a barcoded sequence near a random stretch of DNA, comprising:
combining DNA with control nucleotide sequences to produce a mixture of nucleic acids, each control nucleotide sequence comprising, a primer binding site, a variable nucleotide sequence, and a first recombinase recognition site downstream from the primer binding site, and the variable nucleotide sequence, thereby barcoding the DNA;
amplifying the mixture of nucleic acids to produce amplified nucleic acids;
isolating a plurality of nucleic acids from the amplified nucleic acids, wherein different nucleic acids of the plurality comprise different variable nucleotide sequences and different DNA fragments;
inserting a second recombinase recognition site into the nucleic acids of the plurality, wherein the second recombinase recognition site is inserted into a location that varies among separate individual nucleic acids of the plurality; and
rearranging the nucleic acids of the plurality, thereby producing the barcoded DNA library.
2. The method of claim 1 , wherein the DNA is fragmented DNA.
3. The method of claim 1 , wherein rearranging the nucleic acids of the plurality comprises incubating the plurality of nucleic acids with a recombinase under conditions that favor intramolecular rearrangement.
4. The method of claim 3 , wherein the recombinase is selected from the group consisting of:
Cre recombinase and the first and second recombinase recognition sites are loxP sites;
Flp recombinase and the first and second recombinase recognition sites are FRT sites; and
Tre recombinase and the first and second recombinase recognition sites are LTR sites.
5. The method of claim 1 , wherein the second recombinase recognition site is conjugated to a label.
6. The method of claim 1 , wherein inserting a second recombinase recognition site comprises incubating the plurality of nucleic acids with a second control nucleic acid and a transposase, the second control nucleic acid comprising the second recombinase recognition site and a transposable element.
7. The method of claim 6 , wherein the transposase is Tn3 transposase, Tn5 transposase, Tn7 transposase, Tn9 transposase, Tn10 transposase, Tn903 transposase, or Tn1681 transposase.
8. The method of claim 1 , wherein each control nucleotide sequence comprises a primer binding sequence upstream from the variable nucleotide sequence.
9. The method of claim 1 , further comprising sequencing the nucleic acids of the plurality.
10. The method of claim 1 , wherein the control nucleotide sequence further comprises an origin of replication.
11. The method of claim 10 , wherein amplifying the mixture of nucleic acids comprises:
transforming host cells with the mixture of nucleic acids; and
culturing the host cells, thereby amplifying the mixture of nucleic acids.
12. The method of claim 10 , wherein amplifying the mixture of nucleic acids comprises rolling circle amplification or multiple displacement amplification.