Method for analysing loss-of-heterozygosity (LoH) following deterministic restriction-site whole genome amplification (DRS-WGA)

1 . A method for analyzing loss-of-heterozygosity (LoH) in at least one sample comprising genomic DNA, the method comprising the steps of:

a. providing the at least one sample comprising genomic DNA;

b. carrying out a deterministic restriction-site whole genome amplification (DRS-WGA) of said genomic DNA;

c. preparing a massively parallel sequencing library from the product of said DRS-WGA;

d. carrying out low-pass whole genome sequencing at a mean coverage depth of <1 on said massively parallel sequencing library;

e. aligning the reads obtained in step d. on a reference genome for said at least one sample;

f. extracting the allelic content at a plurality of loci, wherein said plurality of loci comprises polymorphic loci and/or heterozygous loci;

g. assigning at least one LOH score, each LoH score, corresponding to a genomic window of said reference genome, for said at least one sample as a function of the number of loci with at least two different alleles in said plurality of loci.

2 . The method according to claim 1 , further comprising performing a step of fragment size selection before, during or after said step c. of preparing a massively parallel sequencing library and said step of preparing a massively parallel sequencing library does not include a random fragmentation step.

3 . The method according to claim 2 , wherein said step of size-selection retains fragments in the range from 100 to 800 base pairs.

4 . The method according to claim 3 , wherein said step of size selection retains fragments in the range from 300 to 450 base pairs.

5 . The method according to claim 3 , wherein the peak of fragments retained in said step of size-selection is centered on a base pair range from 150 bp to 600 bp.

6 . The method according to claim 5 , wherein said step of size selection step retains fragments in the range 425-575 base pairs.

7 . The method according to claim 1 , wherein said at least one genomic window has a constant width in base pairs.

8 . The method according to claim 7 , wherein each LoH score corresponds to the number of heterozygous loci in said genomic window.

9 . The method according to claim 8 , wherein, if said LoH score passes a threshold for a genomic window, said genomic window is called as being in LoH.

10 . The method according to claim 9 , further comprising a step of assigning an LoH status to at least one genomic region if each LoH score for each genomic window comprised in that region passes said threshold.

11 . The method according to claim 10 , wherein said at least one genomic region comprises a tumor suppressor gene.

12 . The method according to claim 11 , wherein said tumor suppressor gene is selected from the group consisting of:

a. BRCA1

b. BRCA2

c. PALB2

d. TP53

e. CDKN2A

f. RB1

g. APC

h. PTEN

i. CDKN1B

j. DMP1

k. NF1

l. AML1

m. EGR1

n. TGFBR1

o. TGFBR2

p. SMAD4.

13 . The method according to claim 9 , further comprising a step of assigning an LoH status to at least one genomic region as a function of the LoH status of genomic windows comprised in that region.

14 . The method according to claim 1 , wherein said at least one genomic window has a constant number of said plurality of loci.

15 . The method according to claim 1 , wherein said at least one genomic window is selected from the group consisting of a chromosome, a chromosome arm, and a segmented copy-number region.

16 . The method according to claim 1 , wherein said plurality of loci comprises polymorphic loci for the reference genome for said at least one sample.

17 . The method according to claim 16 , wherein each LoH score corresponds to the proportion of heterozygous loci with respect to the total number of said polymorphic loci in the at least one genomic window.

18 . The method according to claim 16 , wherein each LoH score corresponds to the p-value of a statistical test.

19 . The method according to claim 18 , wherein said statistical test assesses the significance of over-representation of biallelic loci with respect to sequencing and WGA error rates.

20 . The method according to claim 18 , wherein said statistical test assesses the significance of under-representation of biallelic loci with respect to a control sample.

21 . The method according to claim 20 , wherein said control sample comprises at least one genomic region at main ploidy from said at least one sample.

22 . The method according to claim 20 , wherein said control sample is an at least one normal sample.

23 . The method according to claim 22 , wherein said at least one normal sample is obtained from the same individual under test from which said at least one sample was obtained.

24 . The method according to claim 20 , wherein said control sample is a maternal or paternal sample, for said at least one sample.

25 . The method according to claim 1 , wherein said at least one sample has a purity of at least 50%.

26 . The method according to claim 25 , wherein said at least one sample is a single cell.

27 . The method according to claim 1 , wherein in step d low-pass whole genome sequencing is carried out at a mean coverage depth of <0.05 on said massively parallel sequencing library.

28 . The method according to claim 1 , wherein in step d low-pass whole genome sequencing is carried out at a mean coverage depth of <0.01 on said massively parallel sequencing library.