Genome-wide capture of inter-transposable element segments for genomic sequence analysis of human DNA samples with microbial contamination

11634,774

16/613667

May 18, 2018

A method for identifying one or more genomic variations in human genomic DNA, comprising employment of Alu, MIR and SVA sequence-based PCR primers and performing an inter-transposable-element (ITE) polymerase chain reaction on the assay mixture to produce an array of amplicons comprising the ITE genomic segments. Also provided the use of the method for identifying one or more genomic variants associated with a trait or a disease.

PharmacoGenetics Limited (Shatin, CN)

PharmacoGenetics Limited (Shatin, HK)

1. A method for identifying one or more genomic variations in human genomic DNA, the method comprising: forming an assay mixture comprising (a) a test sample comprising human genomic DNA; (b) five or more primers, wherein each primer comprises a consensus sequence based on the sequence of a transposable element (TE) or other repeating element found within the human genome, and wherein the TE or other repeating element is not typically found in microbial DNA; (c) free deoxynucleotide triphosphates (dNTPs) comprising adenine (A), cytosine (C), guanine (G), and thymine (T) bases; (d) a thermostable DNA polymerase; and (e) a buffer solution; performing a polymerase chain reaction (PCR) on the assay mixture to produce an array of amplicons comprising inter-transposable element (ITE) genomic segments; sequencing the amplicons using high-throughput DNA sequencing; and comparing sequences of the amplicons to a sequence from the same region of the human genome in a control DNA sample to identify one or more genomic variations between the test sample and the control DNA sample; wherein the assay mixture comprises: (a) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 9; (b) at least six primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 11, and 12; (c) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 8; (d) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 7; (e) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 6; (f) at least six primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 9, and 10; (g) at least ten primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, 11, and 12; or (h) at least eleven primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

2. The method of claim 1 , wherein the one or more genomic variations comprise a single nucleotide polymorphism (SNP), a microsatellite (MST), a germline copy number variation (CNVG), a somatic copy number variation (CNVT), or a combination of any thereof.

3. The method of claim 1 , wherein the test sample comprises human white blood cells, human sputum, human feces, human plasma, human serum, human urine, human saliva, or a combination of any thereof.

4. The method of claim 1 , wherein the test sample comprises human feces obtained from a human subject, wherein the human subject has consumed a diet free of mammalian meat or tissue for at least about 72 hours prior to collection of the test sample.

5. The method of claim 1 , wherein the test sample comprises a sub-microgram quantity of human DNA.

6. The method of claim 1 , wherein the test sample further comprises microbial DNA, plant DNA, or both microbial DNA and plant DNA.

7. The method of claim 1 , wherein the high-throughput DNA sequencing comprises massively parallel sequencing and the massively parallel sequencing produces a massively parallel inter-transposable element (ITE) sequence pattern.

8. The method of claim 1 , wherein the method further comprises transforming data obtained from the sequencing into a computer-readable format prior to comparing the sequences of the amplicons to the sequence of the control DNA sample.

9. A method for identifying one or more genomic variants associated with a trait or a disease, the method comprising: forming a first assay mixture comprising (a) a first test sample comprising human genomic DNA, wherein the first test sample is obtained from a human subject having the trait or the disease; (b) five or more primers, wherein each primer comprises a consensus sequence based on the sequence of a transposable element (TE) or other repeating element found within the human genome, and wherein the TE or other repeating element is not typically found in microbial DNA; (c) free deoxynucleotide triphosphates (dNTPs) comprising adenine (A), cytosine (C), guanine (G), and thymine (T) bases; (d) a thermostable DNA polymerase; and (e) a buffer solution; forming a second assay mixture comprising (a) a second test sample comprising human genomic DNA, wherein the second test sample is obtained from a human subject that does not have the trait or the disease; (b) five or more primers, wherein the sequences of the five or more primers are identical to the sequences of the five or more primers in the first assay mixture; (c) free deoxynucleotide triphosphates (dNTPs) comprising adenine (A), cytosine (C), guanine (G), and thymine (T) bases; (d) a thermostable DNA polymerase; and (e) a buffer solution; performing polymerase chain reactions (PCR) on each of the first and second assay mixtures to produce a first array of amplicons comprising inter-transposable element (ITE) genomic segments from the first test sample and a second array of amplicons comprising inter-transposable element (ITE) genomic segments from the second test sample; and comparing sequences of the first array of amplicons to sequences from the second array of amplicons to identify one or more genomic variations associated with the trait or the disease; wherein the first assay mixture and/or the second assay mixture comprise: (a) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 9; (b) at least six primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 11, and 12; (c) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 8; (d) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 7; (e) at least five primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, and 6; (f) at least six primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 9, and 10; (g) at least ten primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, 11, and 12; or (h) at least eleven primers, wherein the primers comprise the nucleotide sequences of SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

20130143746 June 2013 Xue

Burns, K.H. and Boeke, J. D. Cell 149:740 (May 11, 2012). (Year: 2012). cited by examiner
Burns, K. H., et al., “Human Transposon Tectonics,” Cell, May 11, 2012, pp. 740-752, vol. 149. cited by applicant

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