METHODS TO CAPTURE AND SEQUENCE LARGE FRAGMENTS OF DNA AND DIAGNOSTIC METHODS FOR NEUROMUSCULAR DISEASE

• US 20140274741A1
• Filed: 03/17/2014
• Published: 09/18/2014
• Est. Priority Date: 03/15/2013
• Status: Abandoned Application

• Current Assignee: Translational Genomics Research Institute
• Original Assignee: Translational Genomics Research Institute
• Inventors: Hunter, Jesse M., Baumbach-Reardon, Lisa
• Application Number:
  US14/217,266
• Publication Number:
  US 20140274741A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material

Abstract

The present invention provides methods of sequencing a large fragment of DNA by hybridizing a set of specifically designed probes to the DNA, shearing the DNA, and sequencing the DNA with Next Generation Sequencing. The probes are designed to target genes of interest at intervals to allow the capture of relatively large DNA fragments. The present invention also provides methods of diagnosing a neuromuscular disease (NMD) comprising detecting mutations in one or more of SCML2, CHRND, OFD1, DYNC1H1, COL6A3, EMD, ARHGAP4, FLNA, MID1IP1, MID1, and CFP.

Claims

1. A method of sequencing a large fragment of DNA, the method comprising:

a) isolating genomic DNA from a biological sample;

b) hybridizing the genomic DNA with a set of probes to form genomic DNA-probe complexes, wherein the set of probes targets sequences across the large fragment of DNA at intervals;

c) purifying the genomic DNA from the complexes with affinity chromatography;

d) shearing the genomic DNA to produce small fragments of DNA, wherein the small fragments of DNA comprise coding and non-coding sequences from the large fragment of DNA; and

e) sequencing the small fragments of DNA with Next Generation Sequencing (NGS) to obtain the sequence of the large fragment of DNA.

2. The method of claim 1, wherein the intervals across the large fragment of DNA are about 500 bp to about 5,000 bp.

3. The method of claim 2, wherein the intervals are regular intervals, the regular intervals being about 1,000 bp to about 2,000 bp.

4. The method of claim 3, wherein the regular intervals across the large fragment of DNA are about 2,000 bp.

5. The method of claim 1, wherein the large fragment of DNA is about 2,000 bp to about 50,000 bp in length.

6. The method of claim 1, wherein the set of probes comprises probes of about 80 bp to about 120 bp in length.

7. The method of claim 1, wherein the large fragment of DNA includes at least one gene.

8. The method of claim 7, wherein the at least one gene is associated with a neuromuscular disease (NMD).

9. The method of claim 1, wherein the large fragment of DNA includes at least fifty genes.

10. The method of claim 1, wherein the set of probes comprise an affinity tag selected from the group consisting of biotin and streptavidin.

11. The method of claim 1, wherein shearing the genomic DNA comprises sonication, needle shearing, restriction digest, acoustic shearing, nebulization, point-sink shearing, or passage through a pressure cell of the genomic DNA.

12. The method of claim 11, wherein shearing the genomic DNA comprises sonication.

13. The method of claim 1, further comprising performing end-repair of the genomic DNA after isolating the genomic DNA, wherein the end-repair prevents overhanging, single-stranded DNA from interfering with the hybridizing of the genomic DNA with the set of probes.

14. The method of claim 1, wherein the NGS comprises ion semiconductor sequencing, cycle sequencing, pyrosequencing, or sequencing using γ

-phosphate-labeled nucleotides.

15. A method of sequencing a large fragment of DNA, the method comprising:

a) isolating genomic DNA from a biological sample;

b) hybridizing the genomic DNA with a first set of probes to form genomic DNA-probe complexes with a portion of the genomic DNA encoding a pseudogene, wherein the set of probes targets sequences across the pseudogene at intervals;

c) removing the portion of the genomic DNA encoding the pseudogene with affinity chromatography;

d) hybridizing the genomic DNA with a second set of probes to form genomic DNA-probe complexes, wherein the second set of probes targets sequences across the large fragment of DNA at intervals;

e) purifying the genomic DNA from the complexes with affinity chromatography;

f) shearing the genomic DNA to produce small fragments of DNA, wherein the small fragments of DNA comprise coding and non-coding sequences from the large fragment of DNA; and

g) sequencing the small fragments of DNA with Next Generation Sequencing (NGS) to obtain the sequence of the large fragment of DNA.

16. The method of claim 15, wherein the intervals across the pseudogene and/or the large fragment of DNA are about 500 bp to about 5,000 bp.

17. The method of claim 15, wherein the intervals across the pseudogene and/or the large fragment of DNA are regular intervals of about 2,000 bp.

18. The method of claim 15, wherein the large fragment of DNA is about 2,000 bp to about 50,000 bp in length.

19. The method of claim 15, wherein the large fragment of DNA includes at least one gene.

20. The method of claim 19, wherein the pseudogene and the at least one gene share at least 80% sequence homology.

21. A method of diagnosing a neuromuscular disease (NMD) in a subject, the method comprising:

a) obtaining a biological sample from the subject;

b) isolating genomic DNA from the biological sample;

c) sequencing in the genomic DNA at least one gene selected from the group consisting of SCML2, CHRND, OFD1, DYNC1H1, COL6A3, EMD, ARHGAP4, FLNA, MID1IP1, MID1, and CFP; and

d) diagnosing NMD in the subject if there is a mutation in the at least one gene.

22. The method of claim 21, wherein the mutation is selected from the group consisting of ASN76SER in SCML2, MET1VAL start loss in SCML2, ASP161ASN in CHRND, a single DNA base pair frameshift deletion at genomic position chromosome 2 position 233398958 in CHRND, GLU958LYS in OFD1, TRP1208LEU in DYNC1H1, LYS2483GLU in COL6A3, a DNA substitution of G to T at the splice junction of EMD at genomic position chromosome X position 153608155, PRO635LEU in ARHGAP4, VAL584LEU in FLNA, ARG655HIS in FLNA, ASP51ASN in MID1IP1, PRO667LEU in MID1, and CYS337TYR in CFP.