Rare cell analysis using sample splitting and DNA tags
View Patent ↗The present invention provides systems, apparatuses, and methods to detect the presence of fetal cells when mixed with a population of maternal cells in a sample and to test fetal abnormalities, e.g. aneuploidy. The present invention involves labeling regions of genomic DNA in each cell in said mixed sample with different labels wherein each label is specific to each cell and quantifying the labeled regions of genomic DNA from each cell in the mixed sample. More particularly the invention involves quantifying labeled DNA polymorphisms from each cell in the mixed sample.
1. A method for determining a presence or absence of a fetal aneuploidy of a fetus in a maternal blood sample from a woman who is pregnant or who is suspected of being pregnant, the method comprising:
(a) obtaining a mixture of fetal and maternal genomic DNA from the maternal blood sample;
(b) conducting whole genome amplification of the mixture of fetal and maternal genomic DNA of (a) to obtain amplified nucleic acid molecules;
(c) conducting ultra-deep sequencing of the amplified nucleic acid molecules obtained in step (b) to produce sequence data representing the complete genome for analysis, wherein ultra-deep sequencing comprises further amplification of the amplified nucleic acid molecules to produce at least one million copies of individual amplified nucleic acid molecules in parallel;
(d) using the sequence data of (c) to quantify DNA regions of at least one chromosome being tested for aneuploidy and of at least one control chromosome that is presumed to be diploid, wherein the quantifying comprises analyzing the sequence data of (c) using computer executable logic recorded on a computer readable medium and executed by a processor; and
(e) determining the presence or absence of a fetal aneuploidy for the at least one chromosome being tested for aneuploidy from quantification of the chromosomal DNA regions of (d).
2. The method of claim 1 , wherein ultra-deep sequencing comprises sequencing-by-synthesis.
3. The method of claim 2 , wherein sequencing-by-synthesis involves synthesizing nucleic acid strands complementary to the amplified nucleic acid molecules and inferring nucleic acid sequences of the amplified nucleic acid molecules from the complementary synthesized nucleic acid strands.
4. The method of claim 1 , wherein the fetal aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of one or more chromosomes.
5. The method of claim 4 , wherein the one or more chromosomes are sex chromosomes.
6. The method of claim 4 , wherein the fetal aneuploidy comprises trisomy.
7. The method of claim 6 , wherein trisomy comprises trisomy 13, trisomy 18, or trisomy 21.
8. The method of claim 4 , wherein monosomy comprises monosomy X.
9. The method of claim 1 , wherein the at least one chromosome being tested for aneuploidy is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y.
10. The method of claim 1 , wherein each of the amplified nucleic acid molecules generated by whole genome amplification comprises a tag.
11. The method of claim 10 , wherein the tag comprises a sequencing element.
12. The method of claim 11 , wherein the sequencing element is about 4, 6, 8, 10, 18, 20, 28, 36, 46, or 50 nucleotide bases in length.
13. The method of claim 11 , wherein the ultra-deep sequencing comprises sequencing-by-synthesis initiated using sequencing primers complementary to the sequencing element.
14. The method of claim 3 , wherein sequencing-by-synthesis comprises detecting an identity of each nucleotide immediately after or upon incorporation of a labeled nucleotide or nucleotide analog into a growing nucleic acid strand complementary to the amplified nucleic acid molecules.
15. The method of claim 2 , wherein sequencing-by-synthesis generates at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, or at least 500,000 reads per hour.
16. The method of claim 15 , wherein the reads have at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, or at least 150 bases per read.
17. The method of claim 1 , wherein ultra-deep sequencing comprises sequencing by ligation.
18. The method of claim 17 , wherein sequencing by ligation comprises a four-color sequencing by ligation.
19. The method of claim 18 , wherein sequencing by ligation comprises hybridizing an anchor primer to one of four positions on the amplified nucleic acid molecules.
20. The method of claim 17 , wherein sequencing by ligation comprises an enzymatic ligation reaction.
21. The method of claim 1 , wherein the amplified nucleic acid molecules of (b) are mixed with beads such that a single amplified nucleic acid molecule attaches to a bead, and wherein ultra-deep sequencing of (c) comprises further amplification of the amplified nucleic acid molecule attached to a bead to produce the at least one million copies of the amplified nucleic acid molecule attached to each bead.
22. The method of claim 21 , wherein the amplified nucleic acid molecule attached to each bead is amplified by polymerase chain reaction (PCR).
23. The method of claim 22 , wherein the beads are placed into a highly parallel sequencing-by-synthesis machine that generates over 400,000 reads in a single 4 hour run.
24. The method of claim 23 , wherein the at least one chromosome being tested for aneuploidy is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y.