Methods of sample preparation
The present disclosure provides methods, compositions, and kits for methods that can improve techniques nucleic acid analysis, and can allow for more reliable and accurate targeted, multiplexed, high throughput sequencing. The methods, compositions, and kits can be used for sequencing target loci of nucleic acid. The methods, compositions, and kits disclosed herein can be used for assisted de novo targeted sequencing. The methods, compositions, and kits disclosed herein can also be used for library labeling for de novo sequencing and phasing.
1. A method of generating a tagged nucleic acid library, comprising the steps of:
annealing a first oligo population to a library template;
performing library template-directed nucleic acid extension from the annealed first oligo population to produce a population of extension products;
affinity tagging the population of extension products;
terminating the library template-directed nucleic acid extension to produce a population of first extension products of indeterminate length, wherein at least some first extension products comprise at least 200 bp; and
adding a second oligo sequence near the 3′ end of the first extension product such that a tagged library of nucleic acid molecules is generated comprising nucleic acids each independently comprising a first oligo sequence, a template derived nucleic acid sequence of indeterminate length, and a second oligo sequence, wherein the first oligo population originates from a first random oligo population.
2. The method of claim 1 , wherein said terminating the library template-directed nucleic acid extension comprises incorporation of a biotin tagged ddNTP.
3. The method of claim 1 , wherein the affinity tagging comprises incorporating biotin bound to a dideoxy moiety at the 3′ end extension.
4. The method of claim 1 , comprising affinity purifying said first extension product.
5. The method of claim 1 , wherein the library template-directed nucleic acid extension is performed with a DNA polymerase having strand-displacement activity.
6. The method of claim 5 , wherein the DNA polymerase is capable of incorporating a biotin-labeled ddNTP at the 3′ end of an extending nucleic acid.
7. The method of claim 1 , wherein said adding a second oligo sequence near the 3′ end of the first extension product comprises:
annealing a population of oligos comprising said second oligo sequence to said first extension product; and
contacting the composition to a nucleic acid extension cocktail comprising a DNA polymerase having strand-displacement activity to form a second extension product annealed to the first extension product.
8. The method of claim 1 , comprising sequencing at least one member of the tagged library.