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 sequencing library nucleic acid comprising:
a first nucleic acid strand comprising:
a 5′ sequence comprising at least 6 random bases contributed by a first strand donor primer that anneals to a nucleic acid sample sequence with at least one mismatch,
a 3′ sequence comprising a fragment of the nucleic acid sample sequence, wherein the fragment comprises at least 200 bp,
a 3′ terminal end that cannot support strand extension; and
at least one non-nucleic acid affinity tag bound at the 3′ terminal position of the first nucleic acid strand;
a second nucleic acid strand, comprising a second strand oligo comprising at least 6 random bases contributed by a second strand donor primer that anneals to the first nucleic acid strand with at least one mismatch,
wherein the second nucleic acid strand 3′ end is annealed to the first nucleic acid strand at an internal position on the first strand, so as to be configured to prime synthesis complementary to the first strand.
2. The composition sequencing library nucleic acid of claim 1 , wherein the first nucleic acid strand is terminated by incorporation of a biotin-tagged ddNTP at the 3′ terminal position.
3. The composition sequencing library nucleic acid of claim 1 , wherein the affinity tag comprises biotin bound to a dideoxy moiety at the 3′ end of the first nucleic acid strand, and wherein the biotin is bound to a streptavidin moiety.
4. The composition sequencing library nucleic acid of claim 1 , wherein the second strand oligo comprises an adapter sequence region.
5. The sequencing library nucleic acid of claim 1 , wherein the second strand oligo is shorter than the first strand.
6. The sequencing library nucleic acid of claim 1 , wherein the second strand at least partially anneals within the 3′ half of the first strand.
7. The sequencing library nucleic acid of claim 1 , wherein the second strand at least partially anneals within 20 bases of the first strand 3′ end.
8. The sequencing library nucleic acid of claim 1 , wherein the second strand is extended via a DNA polymerase to form a complement of the 5′ end of the first strand.
9. The sequencing library nucleic acid of claim 8 , wherein the DNA polymerase is a DNA polymerase having strand-displacement activity.