Accurate genome sequencing of single cells by single-stranded amplification and sequencing
View Patent ↗Methods and devices for accurate genome sequencing, including sequencing of single cells by single-stranded amplification and sequencing are provided herein.
1. A method of sequencing nucleic acid of a single cell, the method comprising:
isolating a plurality of double-stranded nucleic acids of a single cell, wherein each double-stranded nucleic acid of the plurality comprises a first strand of nucleic acid and a complementary second strand of nucleic acid, and wherein the first strand and the complementary second strand are complementary to each other;
denaturing the double-stranded nucleic acids to form a plurality of single strands;
randomly partitioning the plurality of single strands in a plurality of solutions, wherein the plurality of single strands are not bound to beads or other solid supports during the random partitioning, wherein each of the plurality of solutions is separate from each other solution, wherein there is at least a 95% probability that for each double-stranded nucleic acid, the first strand and complementary second strand are in separate solutions, and wherein the number of strands in each solution is substantially similar; and
sequencing each strand of the plurality of double-stranded nucleic acids.
2. The method of claim 1 , wherein the double-stranded nucleic acids comprise double-stranded nucleic acids of at least one of chromosomes or chromosome fragments.
3. The method of claim 1 , wherein each of the plurality of solutions has a volume of about 10 nl or less.
4. The method of claim 1 , wherein each of the plurality of solutions has a volume of about 0.4 nl or less.
5. The method of claim 1 , wherein each strand is amplified by multiple strand displacement amplification.
6. The method of claim 1 , wherein sequencing each strand comprises amplifying nucleic acids for no more than about 24 hours.
7. The method of claim 1 , wherein sequencing comprises an error rate of less than about 1 in 10 10 .
8. The method of claim 1 , wherein the plurality of solutions is positioned in a microfluidic device.
9. The method of claim 1 , wherein the probability is at least about 98% that for each double-stranded nucleic acid, the first strand and complementary second strand are in separate solutions.
10. The method of claim 1 , further comprising amplifying each strand in the plurality of solutions by no more than 10,000-fold.
11. The method of claim 1 , wherein the plurality of double-stranded nucleic acids or plurality of single strands are not amplified before randomly partitioning the plurality of single strands.
12. The method of claim 1 , wherein the single strands have a length of at least 100 kilobases.
13. The method of claim 1 , wherein the plurality of double-stranded nucleic acids or plurality of single strands are not amplified before randomly partitioning the plurality of single strands, and wherein the single-stranded nucleic acids have a length of at least 100 kilobases.