SK500482021A3 - Method for determining the uncertainty of the degree of placental mosaicism of a sample in non-invasive prenatal screening - Google Patents

Abstract

The present invention relates to non-invasive prenatal screening (NIPS), in particular to methods for determining the uncertainty of the degree of placental mosaicism in a sample obtained from a pregnant subject. In accordance with the present invention, the respective widths of the monosomic, trisomal and tetrazomic mosaicism intervals are used to determine the degree of monosomal, trisomic and tetrazomic (placental) mosaicism. The present invention further relates to methods for determining whether or not a sample is capable of determining the degree of placental mosaicism within a "read-count" approach to NIPS.

Description

SK 50048-2021 A3

The field of technology

The present invention relates to non-invasive prenatal screening (NIPS). The present invention relates in particular to methods for determining the uncertainty of the degree of placental mosaicism in a sample obtained from a pregnant woman. The present invention further relates to methods of determining whether or not a sample is suitable for determining the degree of placental mosaicism. In accordance with the present invention, the relative variance of monosomic, trisomic, and tetrasomic intervals is used to determine the degree of monosomic, trisomic, and tetrasomic (placental) mosaicism in a read-count approach to NIPS.

Current state of the art

The approach of counting chromosomal reads relative to non-invasive prenatal testing (NIPT) in most cases uses whole genome sequencing (WGS) of cell-free DNA (cfDNA) (Lo et al., The Lancet 350, 485-487 (1997)) and a subsequent bioinformatics pipeline (Bayindir et al., Eur. J. Hum. Genet. 23, 1286-1293 (2015); Porreco et al., Am. J. Obstet. Gynecol. 21 1, 365-el (2014); Thung et al., Expert. Rev. Mol. Diagn. 15, 111-124 (2015); US2016224724; EP2981921) which results in the total number of aligned, pre-processed, normalized reads in a chromosome or subset of a chromosome. The number of reads, also referred to herein as "chromosome read representation" or "chromosome representation", is then usually compared to a threshold value derived from euploid pregnancies in most cases. In this way, it is usually decided whether the test sample is aneuploid or euploid. There are several variations of the principle just described (Tamminga et al., Adv. Clin. Chem. 74, 63-102 (2016)), involving different cases of the alignment procedure (Bayindir, loc cit; Chiu et al., Proc. Natl. Acad. Sci. 105, 20458-20463 (2008)), preprocessing (Jensen et al., PloS One 8, e57381 (2013); Chandrananda et al., PloS One 9, e86993 (2014)) and normalization (Sehnert et al., Clin. Chem. 57, 1042-1049 (2011); Zhao et al., Clin. Chem. 61, 608-616 (2015); Yeang et al., Ultrasound Obstet. & Gynecol. 44, 25-30 (2014)). as well as subsequent decision-making (Bayindir, loc cit; Tynan, loc cit; Johansson et al., Sci. Reports 7 (2017); Sikkema-Raddatz et al., Sci. Reports 6, 38359 (2016); Balslev-Harder et al. , Prenat. Diagn. DOI10.1002/pd.5116). Furthermore, it is well known from the NIPT literature that maternal mosaicism or copy number variation (CNV) is one possible source of false positive or false negative findings in NIPT (Bianchi et al., Genetics in Med (2018), 20(9): 910-917; Flowers et al., Prenatal diagnosis (2015), 35(19): 986-989).

Noninvasive prenatal testing (NIPS) is a liquid biopsy of the placenta (Harasim and Wagner, Noninvasive Prenatal Testing (NIPT), 67-82, (2018)). The basic goal of NIPS is to determine the aneuploid status of the placenta (Poot, Molecular Syndromology 6(4), 153-155 (2015)); i.e. deciding whether a chromosome is euploidy or aneuploidy. NIPS classifies the sample into one of these categories: monosomic, disomic, trisomic, or tetrasomic. If the placenta is affected by mosaicism, the conventional NIPS places it in one of the categories (monosomy, disomy, trisomy, tetrasomy) and therefore the placenta is misdiagnosed. A misdiagnosis can be harmful. It is therefore interesting to develop a method to measure the degree of mosaicism, for example placental mosaicism.

The essence of the invention

In accordance with the technical problem that is solved by the present invention, the objectives set out above have been met. The technical problem has been solved by means and methods as described herein, illustrated in the examples and defined in the patent claims.

In one aspect of the present invention, the objective was to measure and quantify the degree of mosaicism of a given sample (e.g. pregnant/maternal; placenta; fetus) using a suitable screening method, e.g. NIPS. In another aspect of the present invention, the aim was to quantify the uncertainty of determining the degree of mosaicism, e.g. of placental mosaicism, in a suitable screening method such as NIPS. In further aspects of the present invention, the objectives include providing a quality control (QC) criterion for determining the degree of (preferably fetal) mosaicism, for example placental mosaicism, in a suitable screening method, such as NIPS; determining the suitability of a given sample (e.g. from a pregnant/maternal subject; placental; fetal) for determining the degree of (preferably fetal) mosaicism, e.g. placental mosaicism, in an appropriate screening method such as NIPS; or assessing the appropriateness of implementing appropriate screening methods such as NIPS to determine the degree of (preferably fetal) mosaicism (eg placental mosaicism). The NIPS can generally be calibrated as also described herein and provided in the context of the present invention.

SK 50048-2021 A3

Unless otherwise stated herein, when referring to fetal or non-maternal mosaicism, preferably placental mosaicism is meant or determined in accordance with the present invention. The present invention relates to methods for determining the degree of (preferably fetal) monosomic, trisomic and tetrasomic mosaicism, e.g. of placental mosaicism, based on the respective intervals of mosaicism. In accordance with the present invention, relative variance of monosomic, trisomic, and tetrasomic mosaicism intervals is used to determine the degree of monosomic, trisomic, and tetrasomic (eg, placental) mosaicism within a read-counting approach to NIPS. Advantageously, in accordance with the present invention, fetal (eg placental) mosaicism can be determined as described and provided herein. In a preferred embodiment of the present invention, the maternal trisomic and/or monosomic mosaicism is of known degree μ e [0, 1], In a particular embodiment of the present invention, the degree of trisomic and/or monosomic mosaicism of the mother may be 0 (i.e. the mother may be euploid).

In general, the sample for use in the methods according to the present invention, e.g. from a mother or pregnant woman is assumed to be euploid. However, as described herein, the methods of the present invention make it possible to determine whether a sample, e.g. from the mother or a pregnant woman may show mosaicism, e.g. trisomic or tetrasomic.

Accordingly, one aspect of the present invention relates to a method of determining the uncertainty of the degree of trisomic (preferably placental) mosaicism in a sample with maternal trisomic mosaicism of degree μ according to the interval of trisomic placental mosaicism [ymin, y _ma x] with width w = ^max 7min where where • T is the chromosome read representation (or chromosome representation) of the sample, • / is the fetal fraction of the sample.

• t £ _in is the minimum value of the read representation of the chromosome in the set of euploid samples, • ^ŕ max ^is the maximum value of the read representation in the set of euploid samples, and it is assumed that t ' (1 + θ·5 ·μ·[1-/]) , at < tmax (1 + 0.5 μ [1 - /]) + 0.5 t® _ax /.

In accordance with the present invention, the representation of the maternal chromosome may consist of two components: (1 -μ)·/ is the euploid contribution, and 1.5·μ·/ a is trisomic.

In a similar way, the representation of the placental chromosome can include the euploid component (1 - y)·/ and the trisomic one: Ι.57Λ Assuming that the fetal fraction is/ the chromosome representation of the T sample would be t = [(1 - μ) t ^E + 1.5 μ t ^£ ] -(1-/) + [(1 ť) ' t ^E + 1.5 yt ^F ] f.

In accordance with the present invention, 7), 7-., strip can be defined as the set of all (/t) such that the following two conditions are satisfied:

t < (1 + 0.5 μ) t* _ax + 0.5 (y - μ) t* _ax f, t > (1 + 0.5 μ) t® _in + 0.5 (y - μ) t® _in /.

For a mother with trisomic mosaicism of degree μ, and the implementation of NIPS characterized by the spread of t _in , tm _ax , the degree y of placental trisomic mosaicism of a sample with chromosome representation T and fetal fraction / can lie in the interval [γ^η, y _m ax], where ťmin = max 0.2 ľmax = min 1.2 where it is assumed that the sample is such that (/t) satisfies the following conditions:

t ž trnin ( ¹ + °· ⁵ ú) - 0-5 tmin t ^E _ax (1 + 0.5 μ) + 0.5 t£ _ax (1 - μ) /.

SK 50048-2021 A3

In one embodiment of the present invention, the degree of trisomic (preferably placental) mosaicism in a sample with maternal trisomic mosaicism of degree μ is determined by the midpoint of the trisomic (preferably placental) mosaicism interval of the sample, i.e. according to ý = 0.5 (y _m i _n + y _max ).

In another embodiment, the present invention relates to a method for determining whether or not a sample characterized by its lethal fraction f, chromosome read representation T, maternal trisomic mosaicism grade μ, and trisomic mosaicism interval width w, as defined above, meets the conditions for determining the degree of trisomic mosaicism. of (preferably placental) mosaicism with maximum uncertainty ω', comprising determining whether w < w', where if w > w' said sample is not eligible for determining the degree of trisomic mosaicism with maximum uncertainty vr or where if w < w' said sample is capable of determining the degree of trisomic mosaicism, with maximum uncertainty vr'.

In another embodiment, the present invention relates to a method as defined above, wherein in a sample with maternal trisomic mosaicism, the trisomic (preferably placental) mosaicism relates to any autosome or sex chromosome (preferably X).

In accordance with the present invention, the lower and upper bounds of the interval may be more conveniently expressed in terms of z-scores z^ _in , z^ax instead of t^ _in , t^ax· Accordingly, in one embodiment of the present invention, the representation of the reading T of the chromosome replaced by the z-score z where μ ^ζ is an estimate of the distribution of the representation of chromosome reads in a population that is euploid in chromosome, c is an estimate of the extent of the distribution of the representation of chromosome reads in a population that is euploid in chromosome, including the determination of the uncertainty of the degree of trisomic (preferably placental ) of mosaicism in a sample with maternal trisomic mosaicism of degree μ using the trisomic (preferably placental) mosaicism interval [y _min , 7 _max ] with width w = - /min where ľmin = max 0.2 ζ·σ ^ε + μ ^ε + (σ ^ε · ζ ^ε 3Χ + μ ^£ ) · (0.5 μ (/ - 1) - 1)'f-(σ' ¹ · ζ ^ε 3Χ + μ ^Ε ) . Λ ₉ ζ σ ^Ε + μ ^Ε + (σ ^Ε ζ£ _ίη +μ ^Ε ) (0.5 μ (f - 1) - 1) / _max = min 1.2--—g---——---- ------\ f-{σ ^ε · ζ ^Ε _in + μ ^Ε ) where z^ _in is the minimum z-score in the set of samples that are euploid for the chromosome, and z^ _ax is the maximum z-score in the set of samples , which are euploid for the chromosome and it is assumed that z > z ^E _in (1 + 0.5 μ [1 - /]) + 0.5 and < z ^E _ax (1 + 0.5 [μ (1 -/) +/]) + 0.5

In a particular embodiment of the method provided and described herein in accordance with the present invention, μ may be 0.

In another embodiment, particularly when μ is 0, the present invention relates to a method of determining whether or not a sample from a maternal euploid on a chromosome is capable of determining the degree of trisomic (preferably placental) mosaicism on a chromosome with an uncertainty of at most ω' is equivalent to determining whether t tmin + θ·5 w' -f -t ^E _in , (1) t tmax + 0-5 (1 - W ^z ) / (2) (t - tmax) ' (/loď - /lod) ž _in ) (/ where • í is the representation of the chromosome reading of the sample, • /is the fetal fraction of the sample, • t ^E _in is the minimum value of the representation of the chromosome reading in the set of euploid samples, • ^ŕ max í ^e the maximum value of the representation of the chromosome reading in the set of euploid samples, and ( 3) /iS) = 2 ^v max _t ^E .

'•min w'·

SK 50048-2021 A3 /lod = 2 'max ^L min tmin - (1 - ^w ') tmax' + E . fE ^wt max ''min /lod . — (1 — w'·) 'mm \ ^xw J ^l max where if all points (1) to (3) are met, the said sample is not suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w', or where, if not any of the points (1) to (3) are met, the said sample is suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w'.

The present invention further relates to a method for determining the uncertainty of the degree of monosomic (preferably placental) mosaicism in a sample with maternal trisomic mosaicism of degree μ using the interval of monosomic (preferably placental) mosaicism [p _m i _n , p _max ] with width v = p _max -p _m i _n , where

Pmin = max 0, -2 t + 4ίη · (°· ⁵ μ (/ - 1) - 1) f -t ^E .

J 'min. Λ ₉ t + tLx (0.5 ·μ-(/-1)-1)

Pmax = min 1,-2--—7?----------\ J ^max where • í is the representation of the chromosome reading of the sample, • /is the fetal fraction of the sample, • _in is the minimum value of the representation of the chromosome reading in a set of euploid samples, • tmax ^is the maximum value of chromosome read representation in a set of euploid samples, and it is assumed that t tmin ' ( ¹ + °· ⁵ [ú ' (1 - /) - /]). at < tmax (1 + 0.5 μ [1 —/]).

In accordance with the present invention, the representation of the maternal chromosome can consist of two components: (1 -μ)·^ is the euploid contribution and 1.5 μΊ ^! '^ trisomic contribution. Representation of the placental chromosome may include the euploid component (1-p) Y and the monosomic component: 0.5-pt ^E . Assuming that it is a fetal fraction, the representation of the chromosome t of the sample can be t = [(1 - μ) t ^E + 1.5 μ t ^F ] (1 - /) + [(1 - p) t ^E + 0.5 pt ^B ] /.

In accordance with the present invention, the Τ _{μ μ ρ} band can be defined as the set of all (/t) such that the following two conditions are satisfied:

t < (1 + 0.5 μ) t ^E nax - 0.5 (p + μ) t ^E iax /, and > (1 + 0.5 μ) t® _jn - 0.5 (p + μ) t® _jn /.

In a mother with trisomic mosaicism of degree μ, and implementation of NIPS characterized by variance in, tmax. the degree p of the placental monosomic mosaicism of the sample with the representation of the chromosome ta fetal fraction/can lie in the interval [p _min , pmax], where

Pmin = max 0, -2 t + tm _in · (0.5 · μ · (/— 1) — 1) Wiin / t + t ^E

I - _o ' “ ^t max

Pmax = min 1,-2- (0.5 ·μ (/-!)-!) / ' tmax where it is assumed that the sample is such that (/ t) satisfies the conditions:

t ž t® _in (1 + 0.5 μ) - 0.5 t* _in (1 + μ) / and t < (1 + 0.5 μ) - 0.5 t*^ μ /.

In one embodiment, the present invention relates to a method as defined herein, wherein the degree of monosomic (preferably placental) mosaicism in a sample with maternal trisomic mosaicism of degree μ can be determined by the midpoint of the interval of monosomic (preferably placental) mosaicism of the sample, i.e. according to p = 0.5 (p _min + p _max ).

SK 50048-2021 A3

In another embodiment, the present invention relates to a method for determining whether or not a sample characterized by its fetal fraction/reading representation of chromosome t, maternal trisomic mosaicism grade μ and the width of the v interval of monosomic (preferably placental) mosaicism as defined above is capable of determining the degree of monosomic of mosaicism with maximum uncertainty v' including determining whether v < v', where if v > v' said sample is not qualified to determine the degree of monosomic mosaicism with maximum uncertainty v' or where if v < v' said sample is eligible for determination degree of monosomic mosaicism with maximum uncertainty v'.

In another embodiment, the present invention relates to a method as defined herein, wherein monosomic (preferably placental) mosaicism refers to any autosome or sex chromosome (preferably X).

In accordance with the present invention, the lower and upper limits of the interval may be more conveniently expressed in terms of z-score z ^£ in, z ^£ ax instead of t ^£ in, t ^£ ax. Accordingly, in one embodiment of the present invention, the chromosome read representation t can be replaced by the z-score z, t - μ ^£ z =--r~ σ ^Ε where μ ^Ε is an estimate of the location distribution of the chromosome read representation in a population that is euploid in a chromosome, σ ⁶ is an estimate of the extent of the distribution of the representation of a chromosome read in a population that is euploid in a chromosome, involving the determination of the uncertainty of the degree of monosomic (preferably placental) mosaicism in a sample with maternal trisomic mosaicism of the degree μ according to the interval [p _m i _n , /„J of monosomic (preferably placental) mosaicism with width V Pmax Píniu, where

Pmin = max 0.-2 ζ·σ ^Ε + μ ^Ε + (σ ^Ε z ^£ _in + μ ^£ ) · (0.5 μ (/ - 1) - 1/ f4ίη + ú*) . Λ „ z σ ^£ + μ ^£ + (σ ^Γ Zmax + μ^) (0.5 ·μ (/- 1) - 1)

Pmax min I 1, 2 \ f (σ ^Ε z ^E _lax + μ ^Δ ) where z^ _in is the minimum z-score in the set of samples that are euploid for the chromosome, and az^ax is the maximum z-score in the set of samples that are euploid for the chromosome, and it is assumed that z > z ^£ _in (1 + 0.5 [μ (1 - /] - /) + 0.5 _and z < ζ^ _χ (1 + 0.5 μ [1 - /]) + 0.5

In a particular embodiment of the method provided and described in accordance with the present invention, μ may be 0.

In another embodiment, especially if //is 0, the present invention relates to a method of determining whether a sample is suitable for determining the degree of monosomic mosaicism or not suitable for determining the degree of monosomic mosaicism with the maximum uncertainty v'is equivalent to determining whether t >à ^£ _in -0.5-(1-^)-/-^,(4) t tmax - 0.5 v' / (5) (t - £in) ' - /lod) > (í _/L M _D - 4in) ' { f &(6) where • í is the representation of the chromosome reading of the sample, • /is the fetal fraction of the sample, • ^{lmin is} the minimum value of the representation of the chromosome reading in the set of euploid samples, • tmax is the maximum value of the representation of the chromosome reading in the set of euploid samples, and — t ^E

..car _ 'max hmn

f M _ n _____^max 'min_____ ^z — ri — ν'Ί ' ^L max ^v ) ^- ' 7lod) ^max

SK 50048-2021 A3 pncom if all points (4) to (6) are met, the said sample is not suitable for determining the degree of monosomic mosaicism with the maximum uncertainty v', or where, if any of the points (4) to ( 6), the mentioned sample is suitable for determining the degree of monosomic mosaicism with the maximum uncertainty in

The present invention further relates to the method of determining the uncertainty of the degree of trisomic (preferably placental) mosaicism in a sample with maternal monosomic mosaicism of degree μ' according to the interval [y ' _min , γ max] of trisomic placental mosaicism with a width w = / _max - /„„n where , £-^-(0.5-/-(/-1) + 1) y min = max I 0,2--—j----------\ J ^max , ,/ £-^( 0.5-/(/-1) + 1)

Y max = min 1.2--—---------'min where • í is the representation of the chromosome reading of the sample, • / is the fetal fraction of the sample.

• _in is the minimum value of the representation of the chromosome reading in the set of ^euploid samples • max ^is the maximum value of the representation of the chromosome reading in the set of euploid samples, and it is assumed that t > tL _n (1 - 0.5 / [1 -/]), at tmax (1 - 0-5 [μ' -(1+ μ') /])

In accordance with the present invention, the maternal chromosome representation may consist of two components: (1 -/// is the euploid contribution, and 0.5-/'/ is the monosomic contribution. The placental chromosome representation may include the euploid component (1 - y// and the trisomic component: Ι.δ-γ' Ρ. Assuming that the fetal fraction is/ the representation of the chromosome t of the sample can be t = [(1 - μ') t ^E + 0.5 μ' t ^E ] -(1-/) + [(1 - /) t ^E + 1.5 y' t ^E ] /.

In accordance with the present invention, Μ _μ ·Τ _γ · the strip can be defined as the set of all (/t) such that the following two conditions are satisfied:

t < (1 - 0.5 μ') t ^E ax + 0.5 (y' + μ') t ^E ax /, t > (1 - 0.5 μ') t ^E in + 0.5 (y' + μ') t ^E in /.

For a mother with monosomic mosaicism of degree μ', and NIPS implementation characterized by variance t^in, tmax, the degree γ' of placental trisomic mosaicism of the sample with the representation of chromosome ta fetal fraction lies in the interval [y' _m i _n , y max], where , £ -^-(0.5-/-(/-1) + 1)^

Y min = max ( 0.2--—------------I, \ J ^max / /max = min 1.2 tt£ _in -(0.5-/-(/-1 ) + 1).E 'min where it is assumed that the sample is such that (f, t) satisfies the conditions:

t > tm _in (1 - 0.5 /) + 0.5 t ^E in / / and t < (1 - 0.5 /) + 0.5 t ^E ax (g' + 1) f.

In one embodiment, the present invention further relates to a method as defined herein, where the degree of trisomic (preferably placental) mosaicism in a sample with maternal monosomic mosaicism of degree μ' is determined by the midpoint of the trisomic (preferably placental) mosaicism interval of the sample, i.e. according to γ ' = 0.5 (y' _min + Y' _max ).

In another embodiment, the present invention relates to a method for determining whether or not a sample characterized by its fetal fraction/chromosome read representation t, maternal monosomic mosaicism grade/' and trisomic mosaicism interval width w as defined herein is eligible for trisomic grade determination. of (preferably placental) mosaicism with a maximum uncertainty of w'", including determining whether w < w', while if w > w the given sample is not suitable for determining the degree of trisomic mosaicism with a maximum uncertainty of w'", or if w" < w' ” the given sample is capable of determining the degree of trisomic mosaicism with the maximum uncertainty w'”.

SK 50048-2021 A3

In another embodiment, the present invention relates to a method as defined herein above, wherein monosomic (preferably placental) mosaicism refers to any autosome or sex chromosome (preferably X).

In accordance with the present invention, the lower and upper limits of the interval may conveniently be expressed in z-score ^z min, ^z max instead of in, t^x· Accordingly, in one embodiment of the present invention, the representation of the t chromosome read is replaced by a z-score zz = ^, σ ^Ε where p ^E is an estimate of the location distribution of the chromosome read representation in a population that is euploid in chromosome, ďjc an estimate of the extent of the distribution of representation of a chromosome read in a population that is euploid in chromosome, including the determination of the uncertainty of the degree of trisomic (preferably placental) of mosaicism in a sample with maternal monosomic mosaicism of degree //' according to the interval of trisomic (preferably placental) mosaicism [/ _m i _n , / _ma x] with wide w” = y max y min, where / ζ·σ ^ε + μ ^ε -( σ ^ε · z^ _ax + μ ^Ε ) · (0.5 μ' (f - 1) + 1) Ζ·(σ ^£ ·ζ^χ + μ ^£ ) , _ . Λ ₉ ζ·σ ^ε + μ ^ε ~[σ ^ε · ζ ^Ε _ίη +μ ^Ε ) · (0.5 · μ' · (/ - 1) + 1) ť max mm^í, 2 · / - (σ* ^ f _lin + μ*) where ζ^™ is the minimum z-score in the set of samples that are euploid for the chromosome, and az^ _ax is the maximum z-score in the set of samples that are euploid for the chromosome, and provided that z > z ^E _in (1 - 0.5 μ' [1 - /]) - 0.5 z < Zmax d - 0.5 [μ' - (1 + μ') f]) - 0.5

The present invention further relates to a method for determining the uncertainty of the degree of monosomic (preferably placental) mosaicism in a sample with maternal monosomic mosaicism of degree μ' by means of the interval of monosomic (preferably placental) mosaicism [p'mm, p' _ma x] with width v = p'max- p'min, where

P min

ŕ - t ^E _in (0.5 ·μ'(/-!) + !) / ^ŕ min , . Λ ₉ tt^· (0.5 μ' (/— 1) + 1)

P max min (1,2 . _E \ J ' anax where • í is the representation of the chromosome reading of the sample, • /is the fetal fraction of the sample, • is the minimum value of the representation of the chromosome reading in the euploid sample set, • tmax is the maximum value of the representation of the chromosome reading in set of euploid samples, and it is assumed that ^f tmin ' ( ¹ - °· ⁵ '[M' - (P' ~ 1) ' /]) ^and t < í ^£ ax (1 - 0.5 μ' [1 -/] )

In accordance with the present invention, the maternal representation of the chromosome may be composed of two components: (1-p')-t ^E is the euploid contribution, and G.5p't ^E is the monosomic contribution. The representation of the placental chromosome includes the euploid component (1 -pa monosomic component: 0.5 -p ' t ^E . Assuming that the fetal fraction is/ the representation of the chromosome í of the sample can be t = [(1 - μ') t ^E + 0.5 μ' t ^E ] -(1-/) + [(1 - p') t ^E + 0.5 p' t ^E ] /.

In accordance with the present invention, the Μ _μ ·Μ _ρ · strip can be defined as the set of all (f, t) such that the following two conditions are satisfied:

t < (1 - 0.5 μ') t ^E ax + 0.5 (μ' - p) t ^E ax /, at > (1 - 0.5 μ') t ^E in + 0.5 (μ' - p') t ^E in / .

SK 50048-2021 A3

For a mother with monosomic mosaicism of degree μ', and the implementation of NIPS characterized by the variance _in , tmax , the degree p' of placental monosomic mosaicism of samples with the representation of chromosome ta fetal fraction / can lie in the interval [p' _min , p' _max ], where , Λ ₉ t -t£ _in (0.5-/ -(/-1) + 1)

P min = max 0.-2--—--------min

P max / t — _d n ^{c L} max = min (1,-2--(0,5-/-(/-1) + 1) f ímax where it is assumed that the sample is such that (/ t) satisfies to the conditions:

Í ž tmin ( ¹ - °· ⁵ Å) + 0.5 t£in (/-!)/ and t < t ^E ax (1 - 0.5 μ') + 0.5 ŕ/, _x μ' f.

In one embodiment, the present invention further relates to the method as defined herein above, where the degree of monosomic (preferably placental) mosaicism in a sample with maternal monosomic mosaicism of degree μ' is determined by the midpoint of the interval of monosomic (preferably placental) mosaicism of the sample, ie-p'= 0.5 ( _p'min + _p'max ).

In another embodiment, the present invention relates to a method for determining whether or not a sample characterized by its fetal fraction/chromosome read representation t, maternal monosomic mosaicism grade μ' and monosomic (preferably placental) mosaicism interval width v as defined above is eligible for determination degree of monosomic mosaicism with maximum uncertainty v', including determining whether v < v'”, where if v > v' the specified sample is not eligible for determining the degree of monosomic mosaicism with maximum uncertainty v', or where if v < v' the specified sample is capable of determining the degree of monosomic mosaicism with the maximum uncertainty v'.

In another embodiment, the present invention relates to a method as defined herein, wherein monosomic (preferably placental) mosaicism refers to any autosome or sex chromosome (preferably X).

In accordance with the present invention, the lower and upper limits of the interval may conveniently be expressed in terms of z-scores z^ _in , z^ax instead of _in , t^ax- In accordance with one embodiment of the present invention, the representation of chromosome í reading is replaced by the z-score z where μ ^Ε is an estimate of the location of the distribution of chromosome read representation in a population that is euploid in chromosome, /is an estimate of the extent of the distribution of representation of chromosome reads in a population that is euploid in chromosome, including determining the uncertainty of the degree of monosomic (preferably placental) mosaicism in a sample with by maternal monosomic mosaicism of degree μ' through the interval of monosomic (preferably placental) mosaicism [p min, P max] with width in P max P min, where

P'min = max 0, -2

P max

ζ·σ ^Ε + μ ^Ε -(σ ^Ε · z£in + μ ^£ ) · (0.5 - /-((-1) + 1/ / (σ ^£ z® in + μ ^£ ) z σ ^Ε + μ ^£ - (σ ^£ ζ^+μ ⁵ ) (0.5 μ' (/ - 1) + 1) /(/ζ^ + μ^) where ζ^™ is the minimum z-score in the set of samples that are euploid for the chromosome, az^ax is the maximum z-score in the set of samples that are euploid for a chromosome, and provided that z > z ^E _min (1 - 0.5[μ' - (μ' - 1) /]) - 0.5 _and

E n nc ' H nc T' í ¹ - /) z — z _m a _X (1 θ·5 μ [1 /]) 0.5

In the context of the present invention, the (maternal) sample can generally be any sample from a pregnant woman (e.g. mammal, preferably human) containing nucleic acid molecules (e.g. DNA or RNA, preferably DNA) or their fragments or segments, e.g. of placental or fetal origin (eg circulating fetal DNA or cfDNA). Examples may include, but are not limited to, blood, especially plasma or serum (preferably plasma), preferably obtained from the placenta. The preparation of serum or plasma from a maternal blood sample can be

SK 50048-2021 A3 to be carried out by standard techniques known in the field. The serum or plasma can then be subjected to nucleic acid extraction. Possible methods include, but are not limited to, the controlled heating method described by Frickhofen and Young (1991), as is known in the art. Another convenient method of extracting serum and plasma is treatment with proteinase K followed by extraction with phenoFchloroform. Nucleic acid extraction methods from serum and plasma, which allow purification of DNA or RNA from larger volumes of maternal sample, increase the amount of fetal nucleic acid material for analysis and thereby improve accuracy. The sequence-based enrichment method could also be applied to maternal semen or plasma for specific enrichment of fetal nucleic acid sequences.

In one embodiment of the present invention, the pregnant subject is a mammal, preferably a female.

In accordance with the present invention, cfDNA circulates in maternal blood and can be measured by several sequencing methods, particularly MPS (massive parallel sequencing) methods known in the art and also described herein. cfDNA also includes nucleic acid molecules (particularly DNA) from the fetus of a pregnant subject (preferably human), also referred to as cell-free fetal DNA (cffDNA) (Barrett et al., PloS One (2011), 6: e25202) and cell-free maternal DNA (cfmDNA ). The amount of cffDNA relative to the total amount of maternal cfDNA in the same sample is also referred to as the fetal fraction (f). Such cffDNA can be present as segments or fragments and sequenced by massively parallel sequencing (MPS) methods known in the art or other suitable sequencing methods (e.g. NGS, high-end or third-generation sequencing) and the resulting reads aligned to the human genome and counted, e.g. . by methods known in the art and described and illustrated herein. In this way, the representation of the reading of the chromosome (t), t. j. of the number of reads that are mapped to a particular chromosome or its subregions and subjected to preprocessing and normalization can be determined in accordance with the present invention. In the context of the present invention, it is possible to determine whether the read representation of a chromosome (e.g. 21, 18, 13, 1, 5 or X) or parts thereof (e.g. 22, 12p, 9p, 18p) is higher or lower compared to normal (ie, euploid or disomic) reference, which would then indicate aneuploidy (eg, tetrasomy, trisomy, or monosomy) of the fetal chromosome set (Norwitz et al., Rev Obstet Gynecol (2013)), 6 (2): 48-62). For example, if the number of specific reads for a chromosome exceeds a threshold value that represents normal (euploid or disomic) chromosome status, the result is reported as positive for trisomy for that chromosome (Norwitz et al., Rev Obstet Gynecol (2013), 6(2) : 48-62). The same is true vice versa if it falls below such a threshold that it is then marked as monosomy positive for that chromosome.

In accordance with the present invention, it is also possible to amplify fetal DNA molecules from a sample. Standard nucleic acid amplification systems can be used, including polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification (NASBA), branched DNA methods, and so on. Preferred amplification methods include PCR. At least one oligonucleotide primer specific for the fetal sequence is typically used for amplification. In general, a nucleic acid can be probed using a sequence-specific probe.

LoD can be determined by methods known in the art and also described herein (eg, Fiorentino et al., Prenat. Diagn. 36, 304-311 (2016). URL http://dx.doi.org/10.1002/pd.4780). For example, the LoD can be determined experimentally as is known in the art and as described herein. The LoD may be different for each respective chromosome for which aneuploidy (eg, monosomy or trisomy) is determined in accordance with the methods of the present invention as described and provided herein. In conventional NIPT methods known in the art, the "no call" sample pool contains all samples that have a fetal fraction/below LoD. Such "no call" samples are then usually ignored and discarded. This means that in such conventional NIPT methods, a sample with a fetal fraction below the LoD is considered a "no-call" and thus ignored and discarded, regardless of its read representation t. Improved methods for determining the suitability of a given sample for testing are described in VEP17203198.

"Circulating cell-free DNA (cfDNA)" as used herein comprises (fragments of) nucleic acid molecules (DNA, RNA; preferably DNA) of various lengths that are not organized in the nuclei of cells and are not separated by a membrane from the external medium, but which they are located in a liquid medium within a given subject (preferably a mammal, more preferably a human), such as urine, plasma or serum (preferably plasma). cfDNA can be found in samples from a pregnant subject (preferably a mammal, preferably a human), e.g. from maternal plasma samples. Methods of measuring and counting nucleic acid molecules (especially DNA) in samples (plasma), especially readings from cfDNA as described herein, are obvious to a person skilled in the art. For example, MPS (massively parallel sequencing) methods known in the art, and also described and illustrated herein by way of example, enable the rapid enumeration of myriads of nucleic acid molecules (especially DNA), e.g. reads as described herein (Lo, Open Biol. (2012), 2(6): doi 10.1098/rsob. 120086). Other methods for detecting cfDNA, cffDNA, or cfmDNA are known in the art and are described, for example, in Nigam et al., JIMSA (2012), 25: 119-200.

"Circulating cell-free fetal DNA (cffDNA)" as used herein comprises cfDNA fragments of fetal origin in a given sample of the pregnant mother's fetus (preferably mammalian, more preferably human), preferably cfDNA fragments of fetal origin in the plasma of the pregnant woman. Methods for measuring and counting nucleic acid molecules (especially DNA) in samples (plasma), especially readings from cffDNA, as described are apparent to one skilled in the art.

SK 50048-2021 A3 in this text. For example, MPS (massive parallel sequencing) methods known in the art, and also described and illustrated herein by way of example, enable the rapid enumeration of myriad nucleic acid molecules (especially DNA), e.g. reads as described in (Lo, OpenBiol. (2012), 2(6): doi 10.1098/rsob. 120086).

"Circulating cell-free maternal DNA (cfmDNA)" or "maternal cfDNA" as used herein comprises cfDNA fragments of maternal origin in a given sample of a pregnant mother, preferably cfDNA fragments of maternal origin in the plasma of a pregnant woman. An experienced practitioner is familiar with methods for measuring and counting nucleic acid molecules (especially DNA) in samples (plasma), reading cfDNA, cfmDNA and cffDNA, as described herein. For example, MPS (massively parallel sequencing) methods known in the art and also described and exemplified herein allow rapid counting of millions and billions of nucleic acid molecules (especially DNA), e.g. reads as described in (Lo, Open Biol. (2012), 2(6): doi 10.1098/rsob. 120086).

Fetal fraction (/)” as used herein means the amount of cffDNA relative to the total amount of maternal cfDNA in the same sample (expressed as a percentage). Fetal fraction (also known as fetal DNA fractional concentration) can be measured by means and methods known in the art. Fetal fraction can be measured e.g. in placental samples or in maternal blood plasma samples from pregnant women, while a specific signal originating from the Y chromosome can be used to derive fractions of fetal DNA in pregnancies with male fetuses (Chiu et al., BMJ (2011), 342: c7401; Hudecova et al. col., PLoS One (2014), 9: e88484; also known as the "ffY method"). However, this may not be appropriate for pregnancies with female fetuses, where alternative approaches such as targeted sequencing, e.g. SNPs (single nucleotide polymorphisms) can be used to calculate the proportion of specific fetal alleles relative to shared alleles to infer the fetal DNA fraction. However, this approach requires prior knowledge of the fetal genotype (Liao et al., Clin Chem (2011), 57: 91-101). Other methods different from the SNP approach are known in the art and are described, inter alia, in Lo et al., Sci Transl Med (2010), 2: 61ra91; Yu et al., PNAS (2014), 1 1 1: 8583-8588. Additional methods for the determination of fetal DNA fractions in maternal blood plasma samples (independent of the determining maternal or fetal genotypes) are described, inter alia, in WO 2017/050244. Other methods are known in the art and are also described and exemplified herein and include "seqFFY" (Kim et al., Prenat. Diagn. (2015), 35: 810-815) and "seqFFY" as described in examples. In one embodiment of the present invention, fetal fraction (/) is measured using seqFF or seqFFY.

“NIPS”: The read-count approach to NIPS, as used in this text, refers to read count-based NIPS as proposed independently (Fan et al., doi 10.1073/pnas.0808319105, PNAS (2008)) and ( Chiu et al, PNAS 105(51), 20458-20463 (2008)). In accordance with the present invention, modifications of the laboratory protocol, the bioinformatics channel and the NIPS read-count data-analytical procedure, which are easily understood by a person skilled in the art, can also be used. For example, typical steps in NIPS may include: first, the sample is subjected to WGS. The sequence reads are then aligned to the reference human genome. Low-quality and duplicate reads are then filtered out. Aligned reads are binned and normalized. Patches may be applied, including the GC patch. The number of normalized reads in a chromosome or subset of chromosomes is known as the chromosome representation or chromosome read representation. Chromosome representation is one of two quantities that characterize a sample in NIPS. The second is the fetal fraction. The fetal fraction is the relative amount of fetal (more precisely, placental) cfDNA to total (maternal and placental) cfDNA. Several methods of fetal fraction determination have been described in the literature, including the Y-based method, seqFF, and seqFFY, as also described and illustrated herein. NIPS can be further calibrated by generating mono-, di-, tri- and/or tetrasomic bands as described and provided herein.

"Massively parallel sequencing (MRS)" as used herein refers to the simultaneous, massive, parallel sequencing of extremely large numbers of DNA molecules. It is also referred to in the art and in this text as "next generation sequencing", or "NGS" or as "second generation sequencing, (for some sequencing methods also "third generation sequencing"). One skilled in the art will appreciate that sequence data (i.e., reads) can be mapped to a reference (e.g., human) genome using bioinformatics tools known in the art and also described and exemplified herein (e.g., Bowtie2 , as described in Langmead et al., Nat Methods (2012), 9: 357-359). Unmapped, duplicate, low-quality reads can be filtered out by suitable tools known in the art. Reads mapped (i.e., aligned) to a particular chromosome (eg, 21, 18, 13, 5, 1, X) or parts thereof (eg, 22, 12p, 9p, 18p) can be counted, and the number of reads can then be compared to the number of reads in the chromosome of one or more samples that are chromosomally euploid (Norwitz et al., Rev Obstet Gynecol (2013), 6(2): 48-62). Examples of MPS methods include, but are not limited to, Polona sequencing, (pyro)sequencing 454, llumina sequencing, SOLiD sequencing, DNA nanoball sequencing, DEATH sequencing, Nanopore sequencing and others (cf. e.g. WO 9844151; Duncan et al., Diagn Mol Pathol (2017), doi.org/10.1016/B978-0-12-800886-7.00003-0; Voelkerding et al., Clin Chem (2009), 55(4): 641-658; Wheeler et al., Nature (2008), 452( 7189): 872-876; Canard et al., Gene (1994), 148(1): 1-6; Feng et al., MolEcolRes (2015), 16(1): 91-102; Schuster, Nat Methods (2008 ), 51(1): 16-18; WO 06/084132; DRManac et al., Science (2010), 327(5961): 78-81; Porreca, Nat Biotechnol (2010), 28(1): 43- 44; Harris et al. Science (2008), 320(5872): 106-109; Levene et al., Science (2003),

SK 50048-2021 A3

299(5607): 982-686; Eid et al., Science (2009), 323(5910): 133-138; Niedringhaus et al., Anal Chem (2011), 83(12): 4327-4341; Greininger et al., Genome Medicine (2015) 7: 99).

A "read" as used herein refers to the sequence of a segment or fragment of DNA that may, for example, be derived from a particular chromosome (e.g., 21, 18, 13, 5, 1, or X) or parts thereof (e.g., 22, 12 p. (9p, 18p), obtained by sequencing, e.g. MPS methods; cf. Bayindir et al., Eur. J. Hum. Genet. 23, 1286-1293 (2015); Porreco et al., Am. J. Obstet. Gynecol. 211, 365- el (2014); Thung et al., Expert. Rev. Mol. Diagn. 15, 111-124 (2015); US2016224724; EP2981921. In accordance with the method described and provided herein, such readings are typically cfDNA, cfmDNA and cffDNA are obtained by MPS methods known in the art and described herein.

“Chromosome(s) read representation” or “chromosome(s) representation” (used here synonymously unless otherwise noted), as used herein, means the number of reads mapped to a particular chromosome or subregions thereof (also referred to herein as as "(chromosomal) count read") and optionally filtered, preprocessed and normalized. Normalization can be performed as described herein and further referred to, for example, in Bayindir et al., Eur. J. Hum. Genet. 23, 1286-1293 (2015); Porreco et al., Am. J. Obstet. Gynecol. 211, 365-el (2014); Thung et al., Expert. Rev. Mol. Diagn. 15, 111-124 (2015); US2016224724; EP2981921; McCullughacol., PloS One (2014): MatemiT21). Normalization methods are known in the art and are also described and exemplified herein; cf. Example 4. Suitable methods may include, but are not limited to, those described in the Examples herein and those described, for example, in Jensen et al., PloS One (2013), 8: e57381 andKimakol., Prenat. Diagn. (2015), 35: 810-815.

"Limit of detection (LoD)" as used herein means the value of the fetal fraction below which fetal aneuploidy status is not considered detectable (reportable) in conventional NIPT. LoD can be determined, for example, experimentally, as is known in the art, and also as described in (see, e.g., Fiorentino et al., Prenat. Diagn. 36, 304-311 (2016). URL http://dx.doi. org/10.1002/pd.4780). A commonly used value of LoD is about 4%. In accordance with the present invention, each chromosome (eg 13,18,21, X, 1, 5) or parts thereof (eg 22, 12p, 9p, 18p) has its own LoD for monosomy, trisomy. For example, the LoD for trisomy 21 may be about 3 to 4.5%. As another example, the LoD for trisomy 18 may be about 3.5 to 6.5%. As another example, the LoD for trisomy 13 may be about 3.2 to 5.8%. As another example, the LoD for monosomy of chromosome 21 or 18 may be about 3-4%. As another example, the LoD for monosomy of chromosome 13 can be about 3.5 to 4.5%. For the detection of trisomy, the ff _oD can also be reduced by reducing the interval of representation of euploid readings of chromosomes t^ _ax - t^) and/or increasing t£ _ín . The same applies mutatis mutandis to monosomy. The LoD for determining specific chromosomal aneuploidies may also depend on the read normalization method as shown and described herein.

A "no-call sample" as used herein means a sample for which the degree of monosomic, trisomic, and tetrasomic mosaicism cannot be determined with a sufficiently small uncertainty, where the uncertainty is measured by the width of the monosomic, trisomic, and tetrasomic interval. The set of all non-calling samples for a given maximum tolerable width w' of the trisomic mosaicism interval is called the blind spot for determining the degree of trisomic mosaicism with the maximum uncertainty w'. The set of all non-calling samples for a given maximum tolerable width v' of the monosomic mosaicism interval is called the blind spot for determining the degree of monosomic mosaicism with the maximum uncertainty in v'. The set of all non-calling samples for a given maximum tolerable width u' of the tetrasomic interval is called the blind spot for determining the degree of tetrasomic mosaicism with the maximum uncertainty u'.

The term "aneuploidy" as used herein means the presence of an abnormal number (parts) of chromosomes in a cell and includes any type of deviation from a diploid chromosome set, where two homologous copies of each autosome (eg, in human chromosomes 1 to 22) and two allosomes ( sex chromosomes; e.g., in mammals such as humans, two copies of the X chromosome for females and one copy of the X chromosome and one copy of the Y chromosome for males) are present in a cell of a given subject (e.g., fetus; human fetus), where one copy of each of the chromosome is received from the mother and one from the father (for sex chromosomes in mammals such as humans, one X is received from the mother while the other X or Y is received from the individual's father). The term "aneuploidy" may also be used interchangeably herein with the terms "numeric chromosomal abnormality", "numeric chromosomal aberration" or "numeric chromosomal disorder" as known in the art. The term "aneuploidy" as used herein also includes an abnormal number of individual chromosomes, e.g. only one copy of a specific chromosome (ie, monosomy; eg, monosomy of chromosome 13, 18, 21, or X), or three copies of a particular chromosome (ie, trisomy; eg, trisomy of chromosome 13, 18, 21, or X), or four copies of a chromosome or its parts (ie, tetrasomy; eg, tetrasomy of parts of chromosome 22 (cat's eye syndrome), tetrasomy of the short arm (p) of chromosome 12 (12p; Pallister-Killian syndrome), tetrasomy 9p or tetrasomy 18p; or tetrasomy of gonosomes such as XXXX or XXYY). As used herein, the term "aneuploidy" also includes "trisomy," "tetrasomy," or "monosomy." Analogously, the term "euploidy" describes the normal state of the chromosome set, e.g. for mammals such as humans, two copies of each autosome and two sex chromosomes (XX or XY) (i.e., for mammals, diploidy of a set of n = 23 chromosomes (22 autosomes and 1 sex chromosome) or the status of all chromosomes is disomic (in males normally X

SK 50048-2021 A3 and Y are present as sex chromosomes, as will be apparent to one skilled in the art). Similarly, the presence of two copies of a given chromosome is referred to herein and also in the art as "disomic" (eg two copies of chromosomes 13, 18, 21 or X (for X this means XX = female).

Trisomy', as used herein, means that for at least one chromosome, there are three copies of a chromosome present in a cell within a subject's chromosome set (eg, fetal chromosome set). For example, in the context of the present invention, trisomy may include trisomy for one or more chromosomes 21 ("trisomy 21"), 18,13, 9, 8,22 or X, where three copies of at least one (preferably only one) of said chromosomes are present in at least a subset of cells or all cells of a subject (fetus, e.g. human fetus) or trisomy XXY or XYY where three sex chromosomes are present. In one embodiment of the present invention, "trisomy" means three copies of chromosome 21 ("Down syndrome", "trisomy 21"), 18 ("Edwards syndrome", "trisomy 18"), 13 ("Patau syndrome", "trisomy 13") , 8 ("Warkany syndrome 2", "trisomy 8"), X ("Triple X syndrome", "trisomy X") or three sex chromosomes other than XXX, namely XYY ("Jacobs syndrome") or XXY ("Klinefelter syndrome "), preferably trisomy 21, 18, 13 or X, or three sex chromosomes other than XXX, specifically XYY ("Jacobs syndrome") or XXY ("Klinefelter syndrome"). In a particular embodiment of the present invention, the trisomy status of the fetus is trisomy 13, 18, 21 or X.

In the context of the present invention, trisomic mosaicism may generally involve any autosome or X chromosome. In one embodiment of the present invention, trisomic mosaicism involves any of chromosomes 13, 18, 21, or X.

Monosomy', as used herein, means that for at least one chromosome, there is only one copy of the chromosome present in a cell within a subject's chromosome set (eg, fetal chromosome set). Because almost all complete monosomies are lethal (except, e.g., mammalian (human) monosomy for X, where no Y is also present (XO; Tumer's syndrome), the term "monosomy" as used herein also includes "partial monosomy" as is e.g. (in mammals, humans), partial monosomy for chromosome 1 ('lp36 deletion syndrome') or chromosome 5 ('Cri-du-chat syndrome'), where only parts or segments of the respective chromosomes are missing (also known and reported in herein as structural chromosomal aberration or structural chromosomal disorder) in at least a subset of cells or all cells of a subject (a fetus, e.g., a human fetus). That is, the term "monosomy" as used herein includes "partial monosomy" where only one copy of a particular chromosome (autosomal or sex chromosome) is present, or a part or segment of a particular chromosome is missing in at least a subset of cells or all cells of a subject (a fetus, e.g., a human fetus). For example, in the context of the present invention, "monozygous omy” may include monosomy, for example (especially if the subject is a mammal, e.g. human) for one or more chromosomes 21, 18, 13, 1 or 5 or parts thereof, where only one copy of said chromosome is present or part of one of said chromosomes is missing (also "partial monosomy") in a cell or all cells of the subject (fetus , eg human fetus) or XO monosomy where only one sex chromosome (X) is present. In one embodiment of the present invention, "monosomy" means only one copy of chromosome 21, 18, 13 or a missing part or segment of chromosome 1 ("lp36 deletion syndrome", (partial) monosomy 1") or chromosome 5 ("Cri-du-chat syndrome ", "(partial) monosomy 5"), or only one copy of X (and no Y) ("Tumer syndrome", "monosomy X", XO). In a particular embodiment of the present invention, the monosomy status of the fetus is (partial) monosomy 21, 18, 13, 5 or X, or particularly monosomy 21, 18 or 13 (or X).

In the context of the present invention, monosomic mosaicism may refer to any autosome or chromosome X. In one embodiment of the present invention, monosomic mosaicism refers to any chromosome 21, 18, 13, 5 or X, or particularly monosomy 21, 18 or 13 (or X).

"Tetrasomy" as used herein means that for at least one chromosome or portions thereof, there are four copies present in a cell within a subject's chromosome set (eg, fetal chromosome set). This means that four copies of a chromosome or part of it may be present in at least a subset of a subject's cells (i.e. tetrasomy; e.g. tetrasomy of parts of chromosome 22 (cat's eye syndrome), tetrasomy of the short arm (p) of chromosome 12 (12p; Pallister-Killian syndrome ), tetrasomy 9p or tetrasomy 18p; or tetrasomy of gonosomes such as XXXX or XXYY). As used herein, the term "tetrasomy" also includes "partial tetrasomy" for only portions of a given chromosome.

Generally, in the context of the present invention, tetrasomic mosaicism may involve any autosome or chromosome X. In one embodiment of the present invention, tetrasomic mosaicism involves any part of chromosome 22 (cat's eye syndrome), tetrasomy of the short arm (p) of chromosome 12 (12p; Pallister- Killian syndrome), tetrasomy 9p or tetrasomy 18p; or tetrasomies of gonosomes such as XXXX or XXYY.

In the context of the present invention, in a specific embodiment, a sample may not be eligible for determining the degree of monosomic, trisomic, and tetrasomic mosaicism if at least one of the widths of the monosomic, trisomic, or tetrasomic mosaicism intervals, respectively, is greater than or equal to the corresponding maximum allowable width of the respective interval of mosaicism.

SK 50048-2021 A3

Chromosomal mosaicism is the presence of two or more distinct cell lines in an individual (Taylor et al. Human Reproduction Update 20(4), 571-581 (2014)). There are two forms of mosaicism: general mosaicism and limited mosaicism. General mosaicism affects the fetus and the placenta. When mosaicism affects only certain organs (eg brain, placenta, gonads), it is called limited mosaicism. Thus, there is limited placental mosaicism, which affects exclusively the placenta. In limited placental mosaicism, the percentage of trisomic cells may not be the same in all affected areas of the placenta (Canick et al., Prenatal Diagnosis 33(7), 667-674 (2013)). The percentage of trisomic cells may vary in different areas affected by placental mosaicism. Each region, affected by some degree of mosaicism, contributes cfDNA to maternal blood. Because NIPS is based on cfDNA, the screening result refers to the aggregated or average percentage of mosaicism. In other words, NIPS cannot measure the percentage of mosaicism in a specific area of the placenta. NIPS can estimate the average percentage of mosaicism, i.e. j. degree of mosaicism.

"Trisomic mosaicism": If the percentage of p trisomic cells in the maternal blood that are of placental origin is between [0, 100]% and the remaining (/ - p)% of the cells are euploid, the placenta shows grade p trisomic mosaicism. Note that for p = 0%, the placenta is euploid. For p = 100%, the placenta is trisomic.

"Monosomic mosaicism": If the percentage p of monosomic cells in the maternal blood which are of placental origin is between [0, 100]% and the remaining (/ -p)% cells are euploid, the placenta shows monosomic mosaicism of degree p. Note that for p = 0%, the placenta is euploid. For p = 100%, the placenta is monosomic for the corresponding chromosome.

"Tetrasomic mosaicism": If the percentage p of tetrasomic cells in the maternal blood that are of placental origin is between [0, 100]% and the remaining (/ -p)% of the cells are euploid, the placenta shows tetrasomic mosaicism of degree p. Note that for p = 0%, the placenta is euploid. For p = 100%, the placenta is tetrasomic.

As noted above, in the context of the present invention, "mosaicism" as used herein refers to the presence of a distinct population of cells with different genotypes that is developed from a single fertilized cell, particularly to a different ploidy state between different cells. For example, a portion of a subject's cells may contain a monosomic, trisomic, or tetrasomic state for a given chromosome, while another portion of the subject's cells is euploid (diploid) for a given chromosome. Nevertheless, in the context of the present invention, the term "mosaicism" also refers to a condition where a subset or even all (ie, up to 100%) of a subject's cells have a genotype, particularly a ploidy state, deviating from the euploid (diploid) state. For example, in the context of the present invention, the condition where all cells are mono-, tri- or tetrasomic for a given chromosome is also included under the term "mosaicism" as used herein. That is, in accordance with the present invention, monosomic, trisomic, or tetrasomic mosaicism may also be 100% degree (ie, p = 100%, cf. above) of monosomy, trisomy, or terazomy.

In accordance with the present invention, the monosomic mosaicism interval, the trisomic mosaicism interval and the tetrasomic mosaicism interval (with respect to trisomy) quantify the uncertainty of determining the degree of monosomic, trisomic, tetrasomic mosaicism; respectively Intervals can generally be used to define an interval estimate of the degree of monosomic, trisomic and tetrasomic mosaicism; respectively

In the following aspects and embodiments of the present invention, the maternal trisomic. monosomic and tetrasomic degree is 0 and fetal (e.g. placentas) mosaicism is determined.

Accordingly, in one aspect, the present invention relates to a method of determining the uncertainty of the degree of trisomic mosaicism (preferably placental trisomic mosaicism) in a sample using a trisomic mosaicism interval [y _m i _n , y _max ] of width W — ^max ^min, where / t — í no ^c '-max ľmin = max 0, 2 —— \ J '-max — (λ 1 — %in

Km x min I 1.2. _E \ 1 '''min where í is the chromosome read representation of the sample (also called in this text as the chromosome representation), / is the fetal fraction of the sample, _in is the minimum value of the chromosome read representation in the set of euploid samples, _{and x} is the maximum value of the chromosome read representation in a set of euploid samples, and provided that t > _in and preferably that t < (1 + 0.5 -f) · t^ax·

If the degree of trisomic mosaicism is γ, then the chromosome representation t ^Y of the mosaic sample with fetal fraction f is related to the chromosome representation t ^E of the euploid sample according to the formula = (1 - /) t ^E + f [(1 - y) t ^E + 1.5 yt ⁵ ] (A)

SK 50048-2021 A3 where ŕ is the representation of the chromosome reading in the euploid sample.

In formula A, the term in square brackets (ie [(1 - γ) t ^E + 1.5 y /]) includes the euploid contribution (1 - y) t ^E and the trisomic contribution 1.5 γ t ^E . For γ = 1, the degree of trisomic mosaicism would be 100% (ie, all cells show the trisomic state for a given chromosome).

Accordingly, in order to eliminate 100% mosaicism, in one embodiment of the present invention, in formula (A), γ is not 1.

In principle, every euploid mother with an euploid placenta has the same representation of the chromosome t ^E . In accordance with the present invention, with knowledge of the fetal fraction and chromosome representation of a given sample (e.g., pregnant/maternal; placental; fetal subject), the degree of γ trisomic placental mosaicism can be determined with absolute accuracy by solving the formula A for γ, leading to the formula ( B):

/ = 2^. (B)

In one embodiment of the present invention, the degree of trisomic mosaicism in a sample (eg, pregnant/maternal; placenta, fetal subject) is determined by the midpoint of the trisomic mosaicism interval of the sample, ie y = 0.5 (y _min + y _max ).

In principle, the chromosome representation of a euploid mother with a euploid placenta t ^E varies in the interval of values ^min> tmaxl- The main source of variability is the technical variability of methods such as massively parallel sequencing (MPS) or next generation sequencing (NGS) known in the state of the art (also referred to as "first-generation sequencing" (in some sequencing methods also "third-generation sequencing"). Through the above-mentioned formula (A), the variability à is converted to the variability t. Consequently, the degree of mosaicism can only be determined with some uncertainty. The interval t^ _ax ] goes to the interval [ymin, yni _ax ] for the degree of trisomic mosaicism, see Figure 3. Taking this into account, the variability of the formula (B) makes it possible to find the limits of the interval of trisomic mosaicism, which should be í t — t ^E tn ^e ' max /min = max 0.2 _E \ J ^max . Λ " t ~ ŕmin\ /max min I 1.2 " L \ J '''min / where it is assumed that t > and preferably that ŕ < (1 + 0.5 -f) t^ax The present invention also relates to methods dy of determination, whether the sample (e.g. pregnant/maternal; placentas; fetal subject) characterized by its fetal fraction/chromosome read representation t the width of the trisomic mosaicism interval w is or is not capable of determining the degree of trisomic mosaicism with the maximum uncertainty w', including determining whether w <w', if w >w' the said sample does not is capable of determining the degree of trisomic mosaicism with maximum uncertainty w', or where, if w <w', said sample is capable of determining the degree of trisomic mosaicism with maximum uncertainty w'.

In this context, the present invention also provides an embodiment where said method of determining whether or not a sample is eligible for determining the degree of trisomic mosaicism with the maximum uncertainty w' is equivalent to determining whether t^ímin + 0-5-w'- /-t^ _in ,(1) t tmax + 0-5 ' (1 - W') ft£ _ax ,(2) (t - tmax) (/loD “ /lod) ž (f -(3) where í is the chromosome read representation of the sample, /is the fetal fraction of the sample, is the minimum value of the chromosome read representation in the set of euploid samples, _ax is the maximum value of the chromosome read representation in the set of euploid samples, and /S) = 2 ^L max ^c min ^w '· tmin ' /lod = 2 _____^max ^min_____ tmin “ ( ¹ “ ^w ') tmax'

SK 50048-2021 A3 £ _ ^w ^max «-min /lod , — f 1 — ιν ^ζ Ί ' min k ¹ - ^vv J ^L max where if all points (1) to (3) are met, the given pattern is not capable of determining the degree of trisomic mosaicism with the maximum uncertainty w', or where, if any of the points (1) to (3) are not fulfilled, said sample is capable of determining the degree of trisomic mosaicism with the maximum uncertainty w'.

In accordance with the present invention, in one embodiment, if w' is set to 1, then a sample may be considered a sample that does not meet the quality control criterion if its trisomic mosaicism interval is [0,1] For this embodiment of the present invention, for the aneuploidy status of such a sample is considered undeterminable. In a specific embodiment of the present invention, w' is not 1.

In accordance with the present invention, the read representation of chromosome t can be replaced by z-score z

Where μ ^Ε is an estimate of the location of the distribution of chromosome read representation in a population that is euploid in chromosome, is an estimate of the extent of the distribution of representation of a chromosome in a population that is euploid in chromosome, involving the determination of the uncertainty of the degree of trisomic mosaicism in a sample by the interval of trisomic mosaicism [/min, ymax ] with width W — ^max ^min, where y _min = max|0.2-^x ), \ f' t ^z max + _σ £·) /

where z^ _in is the minimum z-score in the set of samples that are euploid for the chromosome, and ^z max ^is the maximum z-score in the set of samples that are euploid for the chromosome, "E and it is assumed that z > _in and preferably , that z < z^ax (1 + 0.5 /) + 0.5 ^ /.

In a specific embodiment of the present invention, to determine the uncertainty of the degree of trisomic mosaicism in a sample as described and provided above, the samples are capable of learning the trisomy status of the fetus on a chromosome according to the following determination:

(a) f<fl _0O (b) ί>(1+0.5th) (c) t < tmax where/is the fetal fraction, t is the representation of the chromosome reading,

Ílod í ^e limit of detection (LoD) for chromosome trisomy, t^ _in is the minimum value of representation of chromosome reads in the set of euploid samples, and ^max í ^e the maximum value of representation of chromosome reads in the set of euploid samples;

where, if all of conditions (a) to (c) are met, said sample is not eligible for the determination of fetal chromosome trisomy status, or where, if any of conditions (a) to (c) are not met, said sample is eligible for determining the trisomy status of the fetus chromosome,

t. j. according to the specific embodiment of the present invention, any of (a) to (c) is not met.

In a more specific embodiment of the present invention to determine the uncertainty of the degree of trisomic mosaicism in a sample, as described and provided above, the samples are suitable for determining the fetal chromosome trisomy status according to the following determination:

(a) / < ff _oD (b) t >(1 + 0.5 f) · t ^E _in (c) t < t ^E ' — 'max where/is the fetal fraction, t is the representation of the chromosome reading, flod í ^e the limit of detection (LoD) for chromosome trisomy, is the minimum value of chromosome read representation in a set of euploid samples, and tmax is the maximum value of chromosome read representation in a set of euploid samples;

where, if all conditions (a) to (c) are met, said sample is not eligible for the determination of fetal chromosome trisomy status, or

SK 50048-2021 A3 whereas, if any of points (a) to (c) is not fulfilled, the said sample is suitable for determining the trisomy status of the fetus of the chromosome, where

2,

t. j. in said more specific embodiment of the present invention, any of (a) to (c) is not met.

The present invention also relates to the method of determining the uncertainty of the degree of monosomic mosaicism in a sample according to the interval of monosomic mosaicism [p _m i _n , p _max ] with width v = p _max -p _mia , where

Pmin = maxi 0.2 tmin ~ ^ŕ f -t ^E

J^min.

/ — f

I _n hnax

Pmax ^— IRÍR I 1, 2 f . fE \ J ^max , where í is the representation of the formula chromosome reading, / is the fetal fraction of the sample,

4 _in is the minimum value of the chromosome read representation in the euploid sample set, t^ax is the maximum chromosome read representation value in the euploid sample set, and it is assumed that, t < t^ _ax and preferably that t > · (1-0.5 · f) .

In one embodiment of the present invention, the degree of monosomic mosaicism in the sample can be determined by the midpoint of the interval of monosomic mosaicism of the sample, ie, according to p = 0.5 (p _min + p _max ).

Regarding monosomic mosaicism, similar considerations lead from the basic equation for the degree p of monosomic mosaicism tP = (1 - /) t ^E + f [(1 - p) t ^E + 0.5 pt ^£ ] (C) to the interval of monosomic mosaicism [pmm , p _max ], given as

Λ - tmin Pmin = max I 0.2 I, \ f ^C min /

I - _n 'UldÄ ' 1 Pmax min I 1 _? 2 I y J ^max / where r is the chromosome read representation of the sample, / is the fetal fraction of the sample, t^n is the minimum value of the chromosome read representation in the set of euploid samples, t^ax is the maximum value of the chromosome read representation in the set of euploid samples, and it is assumed that t < t^ _ax and preferably that t > _in (1-0.5 -f).

For p = 1, the degree of monosomic mosaicism would be 100% (i.e., all cells show monosomic status for a given chromosome). Accordingly, to exclude 100% mosaicism in one embodiment of the contemplated invention, in formula (C), p is not 1.

The present invention further relates to a method of determining whether or not a sample characterized by its fetal fraction/chromosome read representation t and width of the monosomic mosaicism interval v as defined above is eligible or not eligible to determine the degree of monosomic mosaicism with a maximum uncertainty v', comprising determining whether v < v', whereas if v > v' the given sample is not suitable for determining the degree of monosomic mosaicism with the maximum uncertainty v', or whereas, if v < v' the given sample is suitable for determining the degree of monosomic mosaicism with the maximum uncertainty v'.

In one embodiment of the present invention, said method for determining whether or not a sample is suitable for determining the degree of monosomic mosaicism with the maximum uncertainty v' can be equivalent to determining whether f ímin - °- ⁵ (1 - f fmiiv( t — t _max — 0.5 v · f · f _max ,(5) (t - ^in) ' (/ _L oD - /L™) > - tmin) ' (/ ~ Ά(6)

SK 50048-2021 A3 where r is the representation of the chromosome reading of the sample, /is the fetal fraction of the sample, t^in is the minimum value of the representation of the chromosome reading in the set of euploid samples, _{and x} is the maximum value of the representation of the chromosome reading in the set of euploid samples, and

[E — t ^E cEM _ n max ^mm JLoD “ j . _t E ' ^{17 L} max /ld = 2 fE — t ^E ____^max ^c min____ — íl — ΐ/Ί t ^E · ^u max ^u J ^v min ί _Λ Μ _Β = (1-0.5·/ _ο )·ί^ _χ whereas, if all points (4) to (6) are met, said sample is not suitable for determining the degree of monosomic mosaicism with maximum uncertainty v', or whereas, if any of points (4) to (6) are not met, said sample is capable of determining the degree of monosomic mosaicism with maximum uncertainty v'.

In accordance with the present invention, in one embodiment, if v' is set to 1, then a sample may be considered a sample that does not meet the quality control criterion if its trisomic mosaicism interval is [0,1], For such an embodiment of the present invention, for the aneuploidy status of such a sample is considered undeterminable. In a specific embodiment of the present invention, v' is not 1.

In accordance with the present invention, the chromosome read representation i is replaced by the z-score z where g ^E is an estimate of the location of the distribution of the chromosome read representation in the population that is euploid in the chromosome, Đjc is the estimate of the extent of the distribution of the representation of the chromosome read in the population that is euploid in the chromosome , involving the determination of the uncertainty of the degree of monosomic mosaicism in the sample using the interval of monosomic mosaicism [pmm, p _max ] with a width in Pmax Pmin, where

where z^ _in is the minimum z-score in the set of samples that are euploid for the chromosome, az^ax is the maximum z-score in the set of samples that are euploid for the chromosome "E and it is assumed that z < z^ _ax and preferably that z > z^ _in -(1-0.5-/)-0.5 — f.

In a specific embodiment of the present invention, to determine the uncertainty of the degree of monosomy mosaicism in a sample as described and provided above, the samples are capable of determining the monosomy status of a fetus on a chromosome according to the following determinations:

(a) f<f _L ^M _0D (b) t < (1-0.5-f) t^ _ax ( ^c ) t — ^min where/is the lethal fraction, t is the representation of the chromosome reading, flod í ^e limit of detection (LoD ) for chromosome monosomy, t^in is the minimum value of chromosome read representation in the set of euploid samples, and tmax is the maximum value of chromosome read representation in the set of euploid samples;

whereas, if all of (a) through (c) are met, said sample is ineligible for the determination of lethal monosomy status on a chromosome, or whereas, if any of (a) through (c) is not met, said sample is eligible to determine the state of lethal monosomy of a chromosome,

t. i.e., in the given specific embodiment of the present invention, any of points (a) to (c) is not fulfilled.

SK 50048-2021 A3

In a more specific embodiment of the present invention, upon determining the uncertainty of the degree of monosomic mosaicism in a sample as described and provided above, the samples are capable of determining fetal monosomy chromosome status according to the following determination:

(a) f — Ílod (b) t< (1 -0.5 -f)· t^ _ax ( ^c ) J — ^min where/is the lethal fraction, t is the representation of the chromosome reading,

^Ílod is the limit of detection (LoD) for chromosome monosomy, t^n is the minimum representative value of chromosome reads in the set of euploid samples, and tmax is the maximum representative value of chromosome reads in the set of euploid samples; wherein, if all of (a) to (c) are met, said sample is ineligible for determining the monosomy status of the fetus on the chromosome, or whereas, if any of (a) to (c) are not met, said sample is eligible to determine the monosomy status of the fetus on the chromosome where

t. j. in the said more specific embodiment of the present invention, any of points (a) to (c) is not fulfilled.

The present invention further relates to the method of determining the uncertainty of the degree of tetrasomic mosaicism with respect to trisomy in a sample of an odeuploid mother using the interval of tetrasomic mosaicism [0 _m in, 4/,,,.,-,1 with a width of U ^max ^min, where / í t — \\ ^min — max 0.2 · max í 0, ——j — 1 L xxt ^max //

Z tt ^E · \ _/Ί n · í -i ^v min \ ₄ = 2 · min 1,----=— — 1, max i ' c pE /' \ J '''min Z where í is the representation of reading of the chromosome of the sample, /is the fetal fraction of the sample,

4 _in is the minimum value of chromosome read representation in a set of euploid samples, _ax is the maximum value of chromosome read representation in a set of euploid samples, and it is assumed that t > (1 + 0.5 · f) · t^ _in .

In one embodiment of the present invention, the degree of tetrasomic mosaicism with respect to trisomy in a sample from a euploid mother can be determined by the midpoint of the tetrasomic mosaicism interval of the sample, ie, according to Θ = 0.5 (3 _min + e _max \

In the context of the present invention, with respect to tetrasomic mosaicism, the degree ω of tetrasomic mosaicism with respect to euploidy, the chromosome representation τ of a euploid mother with a mosaic placenta, the chromosome representation / of a euploid mother with a euploid placenta, and the fetal fraction / are related according to t = (I - f) t ^E + f [(1 - ω) t ^E + 2 ω t ^B ], (D)

Considering the variability of /, the formula (D) makes it possible to find the limits awj of the interval of tetrasomic mosaicism as / t — t ^E \ / _Λ ^v 'max l — max 0.---?— ,

IÍLLtl i ' c l. E p x I ' ^max / max = min 1,

where it is assumed that t > (1 + 0.5 · /) · _in

For ω = 1, the degree of tetrasomic mosaicism would be 100% (i.e., all cells show the tetrasomic state for a given chromosome). Accordingly, in order to eliminate 100% mosaicism, in one embodiment of the present invention, in formula (D), ω is not 1.

In the context of the present invention, the degree of tetrasomic mosaicism may also be expressed in relation to the trisomic band. For example, a sample with a degree of tetrasomic mosaicism of 0.6 has 60% of placental cells with four copies of a given chromosome, and 40% of placental cells with two copies of a given chromosome.

SK 50048-2021 A3 of the chromosome. The degree of tetrasomic mosaicism can be expressed relative to the lower limit of the trisomic band (ie line: t = (1 + 0.5/) t^ _in ).

For this sample it would be (2*0.6 - 1), i.e. j. the degree of tetrasomic mosaicism, expressed in terms of trisomy, is 20%. The sample would be 20% above the lower limit of the trisomy band.

In accordance with the present invention and in the context of the given example of a sample that is 20% above the lower limit of the trisomy band, the interval of tetrasomic mosaicism [0 _m i _n , 0 _ma x] can be expressed with respect to trisomy given as:

/ í t — \ \

0 _min — ^max ( 0, 2 - ^max ( θ'”7-- ^— 1 l· \ \ J ^max / / nn - 1 λ ^L min l í

PmaY — 2 · min 1,----?— — 1, max i > r -E í ' \ J ' ^min / where í is the representation of the chromosome reading of the sample, / is the fetal fraction of the sample, is the minimum value of the representation of the chromosome reading in the set of euploid samples, _ax is the maximum value of the chromosome read representation in the set of euploid samples, and provided that t >(1+0.5 - f) · t/ _n .

The present invention further relates to a method for determining whether or not a sample from a euploid mother characterized by her fetal fraction/representation of t chromosome readings and the width u of the tetrasomic mosaicism interval as defined above is capable of determining the degree of tetrasomic mosaicism with a maximum uncertainty u', comprising determining whether u < u', wherein if u > u' said sample is not suitable for determining the degree of tetrasomic mosaicism with maximum uncertainty u, or whereas if u < u' said sample is suitable for determining degree of tetrasomic mosaicism with maximum uncertainty u

In one embodiment of the present invention, said method for determining whether a sample from a euploid mother is capable or not capable of determining the degree of tetrasomic mosaicism with the maximum uncertainty u' can be equivalent to determining whether t > tmin + 0.5 (ď + 1) / Í ^E _in , (7) t<t ^E ax + 0.5-(2-ď)-/-t ^E ax, (8) ^_ ' ^_ ' ⁽⁹⁾ where í is the representation of the chromosome reading of the sample, / is the fetal fraction of the sample _. ^_ ' hnax ^Tetra _______^max hnm______ ^L ° ^D t ^E in-(2-ď)-t ^E lax/2' ///“ = ( ¹ + /ToD “) '^min' while if all points are met ( 7) to (9), the said sample is not suitable for determining the degree of tetrasomic mosaicism with the maximum uncertainty u', or whereas, if any of points (7) to (9) are not fulfilled, the said sample is suitable for determining the degree of tetrasomic mosaicism mosaicism u with maximum uncertainty u'

In one embodiment of the present invention, the read representation of chromosome t can be replaced by z-score z

SK 50048-2021 A3 where μ ^£ , is an estimate of the location of the distribution of chromosome read representation in a population that is euploid in chromosome, / is an estimate of the extent of the distribution of representation of chromosome reads in a population that is euploid in chromosome, including the determination of the uncertainty of the degree of tetrasomic mosaicism in the sample from the euploid mother using the interval of tetrasomic mosaicism [0 _min , 0 _max ] with width u = 0 _max where (/ z — Z ^E \ |

0, 2 · max I 0,---------- ^max _E I — 1 I, \ f /

where z^™ is the minimum z-score in the set of samples that are euploid for the chromosome, az^ _ax is the maximum z-score in the set of samples that are euploid for the chromosome,

E and provided that z > (1 + 0.5 /) z/ _n + 0.5

In the context of the present invention, in a specific embodiment, a sample may not be eligible for determining the degree of monosomic, trisomic, and tetrasomic mosaicism if at least one of the widths of the monosomic, trisomic, or tetrasomic mosaicism intervals, respectively, is greater than or equal to the corresponding maximum allowable width of the respective interval of mosaicism.

Chromosomal mosaicism is the presence of two or more distinct cell lines in an individual (Taylor et al. Human Reproduction Update 20(4), 571-581 (2014)). There are two forms of mosaicism: general mosaicism and limited mosaicism. General mosaicism affects both the fetus and the placenta. When mosaicism affects only certain organs (eg brain, placenta, gonads), it is called limited mosaicism. Thus, there is limited placental mosaicism, which affects the placenta exclusively. In limited placental mosaicism, the percentage of trisomic cells may not be the same in all affected areas of the placenta (Canick et al., Prenatal Diagnosis 33(7), 667-674 (2013)). The percentage of trisomic cells may vary in different areas affected by placental mosaicism. Each region, affected by some degree of mosaicism, contributes cfDNA to maternal blood. Because NIPS is based on cfDNA, the screening result refers to the aggregated or average percentage of mosaicism. In other words, NIPS cannot measure the percentage of mosaicism in a specific area of the placenta. NIPS can estimate the average percentage of mosaicism, i.e. j. degree of mosaicism.

"Trisomic mosaicism": If the percentage of p trisomic cells in the maternal blood that are of placental origin is between [0, 100] % and the remaining (1 -p) % cells are euploid, the placenta shows grade p trisomic mosaicism. Note that prep = 0%, the placenta is euploid. For p = 100%, the placenta is trisomic.

"Monosomic mosaicism": If the percentage p of monosomic cells in the maternal blood which are of placental origin is between [0, 100]% and the remaining (/ -p)% cells are euploid, the placenta shows monosomic mosaicism of degree p. Note that for p = 0%, the placenta is euploid. For p = 100%, the placenta is monosomic for the respective chromosome.

"Tetrasomic mosaicism": If the percentage p of tetrasomic cells in the maternal blood that are of placental origin is between [0, 100]% and the remaining (1 - p)% of the cells are euploid, a placenta showing tetrasomic mosaicism grade p. Note that for p = 0%, the placenta is euploid. For p = 100%, the placenta is tetrasomic.

As noted above, in the context of the present invention, "mosaicism" as used herein refers to the presence of a different population of cells with different genotypes that have evolved from a single fertilized cell, particularly to a different ploidy state between different cells.

For example, a portion of a subject's cells may contain a monosomic, trisomic, or tetrasomic state for a given chromosome, while another portion of the subject's cells is euploid (diploid) for a given chromosome. Nevertheless, in the context of the present invention, the term "mosaicism" also refers to a condition where a subset or even all (i.e., up to 100%) of a subject's cells have a genotype, particularly a ploidy state, deviating from a euploid (diploid) state. For example, in the context of the present invention, the condition where all cells are mono-, tri- or tetrasomic for said chromosome is also included under the term "mosaicism" as used herein. That is, in accordance with the present invention, monosomic, trisomic, or tetrasomic mosaicism may also be 100% degree (ie, p = 100%, cf. above) of monosomy, trisomy, or tetrasomy.

In accordance with the present invention, the monosomic mosaicism interval, the trisomic mosaicism interval, and the tetrasomic mosaicism interval (relative to trisomy) quantify the uncertainty in determining the degree of monosomic, trisomic, tetrasomic mosaicism; respectively Intervals can generally serve to

SK 50048-2021 A3 defining an estimate based on the interval of the degree of monosomic, trisomic and tetrasomic mosaicism; respectively

The estimate of γ based on the interval of the degree of trisomic mosaicism can be defined, for example, as ť θ·5 (Kmin T /max)

As another example in accordance with the present invention, an interval-based estimate of degree p of monosomic mosaicism can be defined as

As another example according to the present invention, an interval estimate of the degree of tetrasomic mosaicism with respect to trisomy is defined as

P 0.5 (p _m in 5 Pmax)·

As another example in accordance with the present invention, an interval-based estimate of the degree Θ of tetrasomic mosaicism with respect to trisomy may be defined as = 0.5 (0 _min + 0 _max ).

In accordance with the present invention, the degree of mosaicism (monosomic, trisomic, tetrasomic) can be determined up to the interval of (monosomic, trisomic, tetrasomic) mosaicism. The width of the (monosomic, trisomic, tetrasomic) interval quantifies the uncertainty of determining the degree of (monosomic, trisomic, tetrasomic) mosaicism. The width w of the interval of trisomic mosaicism can then be w = γ™* ^— ^minThe width of the interval of monosomic mosaicism can then be = p _max -p _m i _n . The width u of the tetrasomic mosaicism interval can then be u = 0 _max - 0 _min .

A sample capable of determining the degree of trisomic mosaicism if the width w of the trisomic mosaicism interval is sufficiently small; t. j. if it is less than or equal to the predetermined value w', which expresses the maximum tolerable uncertainty in determining the degree of trisomic mosaicism. Similarly, in accordance with the present invention, a sample is capable of determining the degree of monosomic mosaicism if the width in the interval of monosomic mosaicism is sufficiently small; t. j. if it is less than or equal to the predetermined value v', which expresses the maximum permissible uncertainty in determining the degree of monosomic mosaicism. Finally, a sample is suitable for determining the degree of tetrasomic mosaicism if the width at the interval of tetrasomic mosaicism is sufficiently small; t. j. if it is less than or equal to the predetermined value u', which expresses the maximum permissible uncertainty in determining the degree of tetrasomic mosaicism.

In the context of the present invention, the sample meets the QC criteria, akv < v', aw < w', and u < u'.

In accordance with the present invention, a non-invasive prenatal screening system (NIPS) can be used to measure and quantify the degree of mosaicism (eg, for tri-, mono-, or tetrasomy) of a given sample (eg, pregnant/maternal; placenta; fetal subject). To this end, in specific embodiments of the present invention, calibration of the NIPS system as described herein may be contemplated.

In one aspect, the present invention further relates to a method of calibrating a non-invasive prenatal screening system (NIPS), comprising (a) generating a monosomic, disomic, trisomic and tetrasomic band based on processed sequence data obtained from a plurality of blood samples from a pregnant person containing circulating fetal DNA, while it is known that the karyotype of the fetus of each of the samples is euploid and _in is the minimum value of chromosome representation in the set of euploid samples, tmax is the maximum value of chromosome representation in the set of euploid samples, where

i) the monosomy band is as follows: all pairs (f, t) of the fetal fraction/a representation of the reading of chromosome t such that ί>(ΐ-ο.5·η·ί ^£ _ίη t á (1 — 0.5 f) t^ _ax (ii) the disomy band is as follows: all pairs (f, t) of fetal fraction/a representation of chromosome t such that ŕ > .

^L — ^min ^L — ^l max (iii) the trisomy band is as follows: all pairs (f, t) of the fetal fraction fa a representation of the reading of the chromosome t such that t > (1 + 0.5 /) t ^£ _in t < (1 + 0.5 /) (iv) the tetrasomy band is as follows: all pairs (f, t) of the fetal fraction fa a representation of the reading of chromosome t such that

SK 50048-2021 A3 t > (1 + /) tmin t < (1 + /) t^ _ax (b) determining whether, on the basis of processed sequence data obtained from a number of blood samples from pregnant persons, containing circulating fetal DNA, where it is known the fetal karyotype corresponding to each of the samples, said samples with a monosomic karyotype falling or not falling into the monosomic band, said samples having a trisomic karyotype falling or not falling into the trisomic band, and said samples having a tetrasomic karyotype falling or not falling into the terasomic band as generated in step (a ) whereas, if samples (b) with a monosomic karyotype fall in the monosomic band, samples (b) with a trisomic karyotype fall in the trisomic band, samples (b) with a tetrasomic karyotype fall in the tetrasomic band, the NIPS system is well calibrated;

whereas, if some of the samples (b) with a monosomic karyotype fall outside the monosomic band, or some of the samples (b) with a trisomic karyotype fall outside the trisomic band, or some of the samples (b) with a tetrasomic karyotype fall outside the tetrasomic band was generated in step (a), the NIPS system is not well calibrated.

The present invention further relates to the items listed below in the context of the methods described and provided herein:

1. Method for determining the uncertainty of the degree of trisomic mosaicism in a sample using the interval of trisomic mosaicism [7 _min , /max] with width W — ^max /min, where / t — t ^E \ ~ _n ''max 1 /min = max 0, 2 ——£— I, \ J ^max /

I 1 9 ^min j /max = min I 1.2 _E L \ J ^C min / where

- í is the representation of the chromosome reading of the sample,

- /is the fetal fraction of the sample,

- _in is the minimum value of the chromosome reading representation in the set of euploid samples, - tmax is the maximum value of the chromosome reading representation in the set of euploid samples, and it is assumed that t > _in and preferably that t < (1 + 0.5 · f) t^ _ax .

2. Method according to point 1, where the degree of trisomic mosaicism in the sample is determined by the midpoint of the trisomic mosaicism interval of the sample, ie according to γ = 0.5 (y _min + y _max ).

3. A method for determining whether or not a sample characterized by its fetal fraction/chromosome read representation t and the width of the trisomic mosaicism interval w as defined in point 1 is capable of determining the degree of trisomic mosaicism with maximum uncertainty w', comprising determining, whether w < w', whereas if w > w' the specified sample is not suitable for determining the degree of trisomic mosaicism with maximum uncertainty w or whereas, if w < w' the specified sample is suitable for determining the degree of trisomic mosaicism with maximum uncertainty w

4. The method according to point 3, where said method for determining whether or not a sample is eligible for determining the degree of trisomic mosaicism with the maximum uncertainty w' is equivalent to determining whether í tmin + 0-5 'W' ft ^E _ín , ( 1) t tmax + 0-5 ' (1 - w') ft^ _ax , (t - (/loD - /lod) (tfT _oD - ŕmin) ' (/ “ /ĽÓd).

where

- í is the representation of the chromosome reading of the sample,

- /is the fetal fraction of the sample,

- _in is the minimum value of the chromosome reading representation in the set of euploid samples, - ^ŕ max is the maximum value of the chromosome reading representation in the set of euploid samples, (2) (3) t ^E — t ^E _ n 'min ^ÍLoD “ W ^f - t ^{E vv} 'min /lod = 2 t ^E — t ^E 'max 'min tmin - Ď - ^w ') tmax'

SK 50048-2021 A3 ,,./ . _r E . _t E _ ^w 'max 'min /loD à ^E . — (I — 11/¼ fE / 'mm x ¹ · ^w z 'max where if all points (1) to (3) are met, the said sample is not suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w', or where , if any one of the points (1) to (3) is not fulfilled, the said sample is suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w'.

5. The method of any one of items 1 to 4, wherein the trisomic mosaicism involves any autosome or X chromosome.

6. The method of any one of items 1 to 5, wherein the read representation of the t chromosome is replaced by the z-score z where μ ^ζ is an estimate of the location of the distribution of the chromosome read representation in the population that is euploid in the chromosome, σ ⁶ is an estimate of the range of the distribution of the chromosome read representation in a population that is euploid in chromosome, involving the determination of the uncertainty of the degree of trisomic mosaicism in a sample using the interval of trisomic mosaicism [y _min , ymax] of width w = ymax - y _m in, where

I n Q ^Z ~ ^Z max I /min = max 0.2--1 \ f ' (Zmax + ^)/ \ J x lllclÄ t, -ZZ

where z^ _in is the minimum z-score in the set of samples that are euploid for the chromosome, and z^ax is the maximum z-score in the set of samples that are euploid for the chromosome, E and it is assumed that z > z^ _in and preferably that z < z^ax (1 + 0.5 f) + 0.5 — f.

7. Method for determining the uncertainty of the degree of monosomic mosaicism in a sample using the interval of monosomic mosaicism [p _min , Pmax] with Width V = p _max -Pmin, where / t ^E - t

I ^c m them

Pmin = max I 0.2 \ J ^min / t ^E — t . 'max ^L

Pmax ^— min I 1.2 £ \ T nnax where - r is the representation of the chromosome reading of the sample, - /is the fetal fraction of the sample, - _in is the minimum value of the representation of the chromosome reading in the set of euploid samples, - ^ŕ max is the maximum value of the representation of the chromosome reading in set of euploid samples, where it is assumed that t < t^ax and preferably that t > 4 _in (1 - 0.5 · f).

8. Method according to point 7, where the degree of monosomic mosaicism in the sample is determined using the midpoint of the interval of monosomic mosaicism of the sample, ie according to p = 0.5 · (p^m + p _ma x).

9. A method for determining whether or not a sample characterized by its fetal fraction/chromosome read representation t and the width of the monosomic mosaicism interval v, as defined in point 7, is capable of determining the degree of monosomic mosaicism with a maximum uncertainty v', comprising determining, or v < v', whereas, if v > v' the given sample is not suitable for determining the degree of monosomic mosaicism with the maximum uncertainty in v', or whereas, if v < v' the given sample is suitable for determining the degree of monosomic mosaicism with the maximum uncertainty in '.

10. The method of item 9, wherein said method for determining whether or not a sample is eligible for determining the degree of monosomic mosaicism with the maximum uncertainty v' is equivalent to determining whether

Í tmin-0-5(4) t < 4ax — 0.5 v' / tmax/ (5)

SK 50048-2021 A3 (t - tfin) (/loD - /a) > - ^min) (/ - /a), where

- r is the representation of the chromosome reading of the sample,

- /is the fetal fraction of the sample,

- _in is the minimum value of the chromosome read representation in the set of euploid samples,

- tmax ^is the maximum value of the chromosome reading representation in the set of euploid samples, and (6) /loD = 2 '•max hmn

In ^f t

E max /ÄD = 2 'max ^c min — íl — t/1' ^v max k ^x J ^u min while if all points (4) to (6) are fulfilled, the given sample is not suitable for determining the degree of monosomic mosaicism with the maximum uncertainty v', or whereas, if any of the points (4) to (6) are not met, the said sample is capable of determining the degree of monosomic mosaicism with the maximum uncertainty of v'.

11. The method of any one of items 7 to 10, wherein the monosomic mosaicism relates to any autosome or X chromosome.

12. The method of any one of items 8 to 11, wherein the read representation of chromosome t is replaced by z-score z

Ζ=^, σ ^Ε where t/ jc is an estimate of the location of the distribution of the chromosome read representation in the population that is euploid in the chromosome, ďjc is the estimate of the extent of the distribution of the representation of the chromosome read in the population that is euploid in the chromosome, including the determination of the uncertainty of the degree of monosomic mosaicism in the sample using of the interval of monosomic mosaicism [p _m i _n , p _max ] with width V Pmax Pmin, where / _n n ^z min ~ ^z |

Pmin = max 0.2-j- , \ f / . ₉ ^z max ~ ^z \

Pmax = min 1, 2, \ / ' ÚL + e) \ jx llldXM x / where z^™ is the minimum z-score in the set of samples that are euploid for the chromosome, and ^z max ^is the maximum z-score in the set of samples , which are euploid for chromosome, "E and it is assumed that z < z ^£ _ax and preferably that z > z^ _in -(1-0.5-/)-0.5--^-/.

13. Method for determining the uncertainty of the degree of tetrasomic mosaicism with respect to trisomy in the sample using the interval of tetrasomic mosaicism [0 _m i _n , 0,,,., ,1 with width u — ^max ^min, where í í t — t ^E \ \ d _min = ^max ( 0. 2 · max ( 0,——j — 1), \ \ í ^max / / / t — t ^E \ ^max 2 · min I 1, „ j 1, where

- í is the representation of the chromosome reading of the sample,

- /is the fetal fraction of the sample,

- _in is the minimum value of the chromosome read representation in the set of euploid samples,

- tmax ^is the maximum value of chromosome reading representation in the set of euploid samples, and it is assumed that t > (1 + 0.5 -f) · _in .

14. Method according to point 13, where the degree of tetrasomic mosaicism with respect to trisomy in the sample is determined by the midpoint of the tetrasomic mosaicism interval of the sample, ie according to Θ = 0.5 · (0 _min + 0max).

SK 50048-2021 A3

15. A method of determining whether a sample characterized by its fetal fraction/chromosome read representation t and the width of the tetrasomic mosaicism interval u, as defined in point 13, is suitable or not suitable for determining the degree of tetrasomic mosaicism, with a maximum uncertainty u', comprising determining whether u < u', whereas if u > u' the specified sample is not suitable for determining the degree of tetrasomic mosaicism with maximum uncertainty uor whereas if u < u' the specified sample is suitable for determining the degree of tetrasomic mosaicism with maximum uncertainty u

16. The method according to point 15, wherein said method for determining whether or not a sample is eligible for determining the degree of tetrasomic mosaicism with the maximum uncertainty u' is equivalent to determining whether t > tmin + 0-5 '(U' + 1) / t,V _n ,(7) t á tmax + 0-5 ' (2 - u') f ^ _ax ,(8) ^_ ' - O/Ľío™ “ fe) ' ^_ ^od)'<

where

- í is the representation of the chromosome reading of the sample,

- /is the fetal fraction of the sample,

- _in is the minimum value of the chromosome read representation in the set of euploid samples,

- tmax ^is the maximum value of chromosome reading representation in the set of euploid samples, at ^E — t ^E ^° ⁰ “ ^z ' íl + u'Vt ^E · -t ^E ' ty™ ⁼ Ď + θ·5 /lod) tmax t ^E — t ^E cTetra _______^max ^c min______ ^/LoD “ = í ¹ +/ĽoD' ^a ) hnin· while if all points (7) to (9) are fulfilled, the given sample is not suitable for determining the degree of tetrasomic mosaicism with maximum uncertainty u', or whereas, if any one of points (7) to (9) is not met, said sample is capable of determining the degree of tetrasomic mosaicism with the maximum uncertainty u'.

17. The method of any one of items 13 to 16, wherein the tetrasomic mosaicism involves any autosome or X chromosome.

18. The method of any one of items 15 to 17, wherein the representation of the chromosome read t is replaced by a z-score of σ ^ε where μ ^£ , is an estimate of the location distribution of the representation of the chromosome read in a population that is euploid in the chromosome, σ ⁶ is an estimate of the range of the distribution representation of a chromosome read in a population that is euploid in chromosome, including the determination of the uncertainty of the degree of tetrasomic mosaicism in a sample according to the interval of tetrasomic mosaicism [0 _m i _n , //,,,,1 with width u Qmax ^min, where (/ Z — Z ^E .\

0, 2 · max I 0,--------- ^max _E | — 1 L \ f Az +/ \ * — //mx ľ. jj/

where is the minimum z-score in the set of samples that are euploid for the chromosome, and ^z max is the maximum z-score in the set of samples that are euploid for the chromosome, "E and it is assumed that z > (1 + 0.5 /) ζ ^ _η + 0.5 —.

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19. The method according to point 3, 9 or 15, whereby the sample is not suitable for determining the degree of monosomic, trisomic and tetrasomic mosaicism, if at least one of the widths of the intervals of monosomic, trisomic or tetrasomic mosaicism is greater than or equal to the corresponding maximum permissible width of the respective mosaicism interval .

20. The method of any of the preceding items, wherein the monosomic, trisomic or tetrasomic mosaicism is 100% of the degree of monosomy, trisomy or terasomy.

21. A method of calibrating a non-invasive prenatal screening (NIPS) system comprising (a) generating a monosomic, disomic, trisomic and tetrasomic band based on processed sequence data obtained from a plurality of blood samples from pregnant individuals containing circulating lethal DNA known to be lethal the karyotype of each of the samples is euploid and

- is the minimum value of chromosome representation in the set of euploid samples,

- tmax is the maximum value of the chromosome representation in a set of euploid samples, where (i) the monosomy band is as follows: all pairs (f, t) of the lethal fraction/a read representation of chromosome t such that t > (1 - 0.5 f) tP _n t < (1 - 0.5 /) (ii) the disomy band is as follows: all pairs (f, t) of the lethal fraction/a read representation of chromosome t such that t >

t < t ^{E L} — ^l max (iii) the trisomy band is as follows: all pairs (f, t) of the lethal fraction/a read representation of chromosome t such that t > (1 + 0.5 /) t < (1 + 0.5 /) t ® _ax (iv) the tetrasomy band is as follows: all pairs (/ t) of the lethal fraction/a read representation of chromosome t such that t > (1 + /) t£ _in t < (1 + /) tmax (b) determining whether based on processed sequence data obtained from a number of blood samples from pregnant persons containing circulating lethal DNA, where the lethal karyotype corresponding to each of the samples is known, said samples with a monosomic karyotype fall or do not fall into the monosomic band, said samples with a trisomic karyotype fall or do not fall into the trisomic band band and said samples with a tetrasomic karyotype fall or do not fall within the tetrasomic band as generated in step (a) whereas, if samples (b) with a monosomic karyotype fall within the monosomic band, samples (b) with a trisomic karyotype fall within the trisomic band, samples ( b) with tetrasomic by karyotype they fall into the tetrasomic band, the NIPS system is well calibrated;

whereas if some of the samples (b) with a monosomic karyotype fall outside the monosomic band, or some of the samples (b) with a trisomic karyotype fall outside the trisomic band, or some of the samples (b) with a tetrasomic karyotype fall outside the tetrasomic band as generated in step (a), the NIPS system is not well calibrated.

It should be noted that, as used in the text, the singular forms "a", "an", and "the" (in the English original) include plural references unless the context clearly indicates otherwise. For example, reference to an "agent" includes one or more such different agents, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that can be modified or substituted for the methods described herein.

Unless otherwise indicated, the term "at least" preceding a series of elements is intended to refer to each element in the series. Those skilled in the art will recognize or be able to detect, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are included in the present invention. Also, the term "at least" preceding a specific amount or number also includes exactly that amount or number, i.e. j. it can also serve as a specification of a specific quantity or number.

The term "and/or", wherever used herein, includes the meaning of "and", "or" and "all or any other combination of elements connected by said term".

SK 50048-2021 A3

The term "about" or "approximately" as used herein means up to 20%, preferably up to 10%, and more preferably up to 5% of a given value or range.

Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprising" "and variations such as "comprising" and "comprising" shall be understood to mean the inclusion of said integer or step or set of integers or steps, but not to the exclusion of any other integer or step or set of integers or steps. As used herein, the term "comprising" may be replaced by the term "comprising" or "comprising" or sometimes when used herein by the term "having."

As used herein, the term "consisting of" excludes any element, step, or ingredient not specified in an element of a claim. As used herein, the term "consisting essentially of" does not exclude materials or steps that do not materially affect the essential and novel characteristics of the claim.

In any case herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced by any of the other two terms.

It should be understood that the present invention is not limited to the particular methodology, protocols and reagents, etc., described herein and as such may vary. The terminology used herein serves only to describe specific embodiments and is not intended to limit the scope of the present invention, which is defined exclusively by the patent claims.

All publications and patents cited in the text of this document (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein shall be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of the prior invention. If material incorporated by reference contradicts or contradicts this document, the document will supersede any such material.

Overview of images on drawings

Figure 1: The blind spot for determining the degree of trisomic mosaicism with a maximum uncertainty of 20% is shown as a quadrilateral with a 135 degree line shading in the (f, t) plane. The sample of Example 1 is shown as a triangle. It is in a blind spot, which means that the degree of trisomic mosaicism for this particular sample cannot be determined with a maximum uncertainty of 20%; rather, the width w of the trisomic mosaicism interval for this sample is greater than 20%.

The sample according to example 2 is shown as a square.

The sample lies outside the blind spot, which means that the width of the trisomic mosaicism interval is less than 20%, and therefore the sample meets the QC criterion (i.e., is eligible to determine the degree of trisomic mosaicism such that the degree of trisomic mosaicism according to the present invention) as estimated by the estimate of γ based on the interval which can be communicated to the patient along with the trisomy mosaicism interval.

Figure 2: Representation values of t chromosome 21 reads and fetal fraction/for 10000 euploid samples (indicated by a solid circle) and 50 trisomy 21 samples (indicated by a square) were artificially constructed. Horizontal lines at t = t£ _ín at = t^ax delimit the band of disomy. The upward sloping lines are the borders of the trisomy band, which is connected to and originates from the disomy band. The straight line connected to has the equation t = (1 + 0.5 · f) · t^. The straight line connected st^ _ax has the equation t = (1 + 0.5 · f) t^ _ax . Because all (except one) trisomy 21 samples lie within the trisomy band, this implementation of NIPS is well calibrated and can be used to reliably determine the degree of trisomy mosaicism in accordance with the present invention.

Figure 3: Every euploid placenta has theoretically the same representation of a chromosome readout. Thus, the degree of trisomic mosaicism can be determined with absolute accuracy by solving the equation (*) for gamma. However, in practice, the value of chromosome reading representation for euploid samples varies in a certain range [ t^ _in , tmaxi due to the technical variability of WGS (MPS, NGS). Variability in euploid samples is transferred through the equation (*) to the variability of the chromosome representation of non-euploid samples and this allows determination of the degree of mosaicism only with finite accuracy. The uncertainty of determining the degree of mosaicism depends on tmax- If (f, t) samples are known, the minimum and maximum values [y _min , γ^] of the degree of trisomic mosaicism can be obtained from the equation (*), taking into account the two extreme values of t^, t^ _ax of chromosome representation in euploid samples.

Figure 4: Trisomic and monosomic blind spots

Blind spot for monosomic and trisomic mosaicism when the maximum tolerable uncertainty v ’ for monosomic mosaicism, w ’ for trisomic mosaicism are set to 0.2, 0.4, 0.8 and 1; respectively The size (area) of the blind spot decreases as the maximum tolerable uncertainty increases. For v' = w' = 1, the blind point restores the triangle of uncertainty for monosomy and the triangle of uncertainty for trisomy (cf. EP 17203198).

Figure 5: Tetra-, tri- and monosomic blind spots

SK 50048-2021 A3

Blind spot for monosomic, trisomic, and tetrasomic mosaicism, for the maximum allowable uncertainty for each mosaicism set to 0.2 (i.e. v ’ = w ’ = u ’ = 0.2).

Figure 6: Monosomic, disomic, trisomic and tetrasomic bands

The disomic (euploid) band is the basic band from which the monosomy, trisomy and tetrasomy bands are derived. The disomic band is defined as all pairs (f, t) of the fetal fraction/a of the chromosome representation t, in which t is in [ tmaxl- The minimum and maximum t^ax values of the chromosome representation can be obtained from a cohort of samples with a confirmed euploid karyotype.

Figure 7: Maternal trisomic mosaicism of known grade and placental trisomic mosaicism (.W

A sample with a z-score of 15.6 and a fetal fraction of 0.1 (red dot) would be considered tetrasomic mosaic if the mother was assumed to be euploid; or she could show 50% to 100% trisomy mosaicism if the mother was known to show 7% trisomy mosaicism. Dashed green lines delineate the T _o 1\ trisomic band (not shown). The band is associated with the conventional [-3,3] disomic T ₀ T ₀ band (not shown). The blue solid lines delineate the To.oiTo.n band. Note that 0.75 is the average of the lower (0.5) and upper (1.0) bounds on γ.

Figure 8: Maternal trisomic mosaicism of known grade and placental trisomic mosaicism (W

A sample with a z-score of 13.2 and a 10% fetal fraction (red dot) could be considered trisomic if the mother was predicted to be euploid, or could be euploid if the mother was known to have 10 % trisomic mosaicism.

Figure 9: Maternal trisomic mosaicism of known grade and placental monosomic mosaicism (TmMr)

A sample with a z-score of 0 and a fetal fraction of 10% (red dot) could be considered a sample indicating a euploid placenta if the mother is known to be euploid, or could indicate a 62% (26% to 99%) monosomic placenta mosaicism if the mother was known to have 7% trisomy mosaicism.

Figure 10: Maternal monosomic mosaicism of known degree and placental trisomic mosaicism (M^Ty)

A sample with a z-score of -6.6 on the X chromosome and a fetal fraction of 10% (red dot) indicates a male gender - because the sample is in the "boy band" (dashed green lines), assuming a healthy maternal karyotype. If 10% maternal X0 mosaicism is assumed, then the sample shows 0% to 36% XXX mosaicism; that means it is compatible with XX. The blue solid line represents the z-score of the mosaic mother and corresponds to the lowest z-score among the XX samples; t. j. - 2.7.

Examples of implementation of the invention

Example 1

Step 1) Massively Parallel Whole Genome Sequencing (MPS), Bioinformatics Processing, Read Mapping, Filtering, Binning, Chromosome Read Representation cfDNA from a blood sample from a pregnant woman was sequenced by MPS. The resulting reads were mapped to the human hgl9 genome using the bowtie2 algorithm. Duplicate and low-quality reads were then filtered out. Subsequently, the reads were stored in a 50 kilobase bin for pairs. The total number of reads in a subset of bins was normalized by the total number of reads in all autosomes. A subset of reservoirs was selected by an internal method. Subgroup selection did not assume fetal fraction information, thus avoiding circularity.

In addition, the subset selection was cross-validated. The resulting read representation (subset) of chromosome 21 was t = 0.0082.

Step 2) Determination of minimum and maximum values of read representation of chromosome 21 in euploid pregnancies.

In the set of samples with confirmed euploidy on chromosome 21, the minimum read representation for chromosome 21 in the same subset of bins as used in step 1 was equal to 0.0078291. The maximum value was 0.0079678.

Step 3) Determination of the fetal fraction

The fetal fraction for pregnancy was estimated by the seqFFY method to be 0.15 (i.e. 15%). The seqFFY method for fetal fraction estimation was performed as follows:

Although there are systematic and substantive differences between several methods for determining fetal fraction in male pregnancies (Grendar et al., Prenat. Diagn. (2017), DO 1 10.1002/pd.5151; vanBeek et al., Prenat. Diagn. 37, 769-773 (2017). DOI 10.1002/pd.5079), ffY method (seqFF comparison according to Kim

SK 50048-2021 A3 col., loc.cit. with the Y-chromosome method according to Hudecova et al., PloS One 9, e88484 (2014) and Chiu et al., BMJ 342, c7401 (2011)) are considered reliable (Peng et al., Int. J. Mol. Sci. 18, 453 (2017)). The second mentioned reference also stated that seqFF tends to underestimate the fetal fraction at the lower end of the values. To alleviate the underestimation, the present inventors have made a modification of seqFF, referred to as seqFFY, which borrows the advantages of both of these methods. For male pregnancies, seqFFY is only the ffY method because it can be even more accurate than seqFF. ffY cannot be used for female pregnancies. However, it is possible to calibrate ffY to seqFF and use the calibration curve to generate an estimate for female pregnancies by predicting its ffY value based on the seqFF value. The calibration curve can be estimated using any of the regression methods; cross-validated smoothing spline (R Core Team. R: Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria (2015). URL https://www.R-project. org/) was used to obtain the calibration curve.

Step 4) Determination of the interval of trisomic mosaicism and degree of trisomic mosaicism Determination of the interval of trisomic mosaicism [y _min , 7ni _ax ] with width w = where / t — t ^E \ ~ _n ^{6 l} max 1 /min = max ( 0, 2 —— _E — L x I ^max / — í Λ o .

fmax min l L 2 _E L \ f ''min Z where í is the representation of the chromosome reading of the sample, / is the fetal fraction of the sample, _in is the minimum value of the representation of the chromosome reading in the set of euploid samples, tmax is the maximum value of the representation of the chromosome reading in the set of euploid samples , at is greater than t^ _in ymaj was [0.389, 0.632], The width w of the interval of trisomic mosaicism was 0.243 and the estimate based on the interval of the degree of trisomic mosaicism is γ = 0.510, ie, 51%.

Step 6) Blind spot for trisomic mosaicism, given w' = 0.2

The same conclusion as in step 5) can be reached by checking formulas (1), (2) and (3):

t Stmin + 0-5 //imm.(Ό t á tmax + 0-5 (1 - W') / t ^£ _ax ,(2) (t - tmax) (/boD “ /ice) - t* _in ) (/ - /'/j,(3) where í is the representation of the chromosome reading of the sample, / is the fetal fraction of the sample, is the minimum value of the representation of the chromosome reading in the set of euploid samples, tmax is the maximum value of the representation of the chromosome reading in the set of euploid samples, and ^E — t ^E · "cr _ ^L max '•min t ^E — t ^E rT _ -____imax ^mm____ ^L ° ^D " ^min - U - *0 t^' _ ^wt max ^min /loD fE . — f 1 — t ^E '

Hnin ^w J ^L max where, if all points (1) to (3) are met, the said sample is not suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w', or where, if any of the points (1) to ( 3), the mentioned sample is capable of determining the degree of trisomic mosaicism with the maximum uncertainty w';

see the triangle in Figure L As stated therein, formula (1) represented as Right Side (RHS) was

0.0079465 which is less than t. Also formula (2) represented as RHS was 0.0084458 which is greater than í. Finally, formula (3) also represented as Left Side (LHS) was 3.136037e-06 which is greater than RHS which was

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-2.022559e-05. The sample fell into the blind spot for trisomic mosaicism with a maximum tolerance of w' = 0.2; see the black triangle in Figure 1.

Example 2

For the next sample, the representation of the chromosome 21 read was t = 0.0079825. And the fetal fraction was / = 0.184. The result of this and the t£ _ín at^x values that characterize a particular implementation of NIPS is [y _m m, ľmax], which is [0.020,0.213], The width w of the trisomic mosaicism interval was 0.193 (ie 19.3%). The interval-based degree of trisomic mosaicism was 0.117 (ie 11.7%). Since the width w was less than the preselected maximum allowable width w' of the trisomic mosaicism interval, the sample is eligible for determining the degree of trisomic mosaicism.

t was greater than t^ _ax , and therefore a monosomic mosaicism interval was not defined for the sample (and an estimate based on the interval of the degree of monosomic mosaicism). The tetrasomic interval of mosaicism did not exist for this sample either.

The sample eligible for trisomy mosaicism grade and the interval-based estimate (11.7%) of the trisomy mosaicism grade along with the interval itself ([0.020, 0.213]) could therefore be reported to the patient.

The fact that the sample is suitable for determining the degree of trisomic mosaicism could also be validated by verifying formulas (1), (2) and (3). The first formula was indeed valid for the sample because the RHS was 0.0079732, which is less than t. The second formula also held because the RHS was 0.0085542, which is greater than t. However, the third formula did not hold because the LHS was 1.990691 e-07, which is not greater than 5.143569e-06. The sample fell outside the blind spot for trisomic mosaicism with a maximum tolerance of w' = 0.2; see the square in Figure 1.

Example 3

000 samples that underwent NIPS were later confirmed to show a euploid karyotype.

Based on this cohort, minimum t^ _in and maximum t^ax values of chromosome 21 read representation in the cohort were calculated. The 50 samples that underwent NIPS were later confirmed to have trisomy 21. This particular implementation of NIPS (which includes a laboratory protocol, a bioinformatics pipeline, and a data analysis procedure) was intended to be used to determine the degree of monosomic, trisomic, and tetrasomic mosaicism. Before applying NIPS for this purpose, it was necessary to check that the NIPS implementation is well calibrated. Here it was a matter of checking whether the trisomic samples lie in the trisomy band. This can be done using the formulas applied to the calibration of the NIPS system as described and provided herein, where _in is the minimum value of chromosome representation in a set of euploid samples, and _ax is the maximum value of chromosome representation in a set of euploid samples: for the monosomy band:

t > (1 — 0.5 /) t ^E _in t < (1 - 0.5 /) tma _X , for the band of disomy:

t > t ^E · ^c — ''min t < t ^E — '-max" for the trisomy band:

t > (1 + 0.5 /) /min t < (1 + 0.5 /) tmax.

for the band of tetrasomy: t > (1 + /) _in t<(i + /)-t ^E _ax , or by visual inspection of the graph of pairs of values (/ t); see Figure 3. Because all (except one) trisomic samples were located in the trisomy band, derived from the disomy band, we could show that the NIPS implementation was well calibrated and therefore could be reliably used to determine the degree of placental mosaicism.

Example 4 (^0.07^0.75): Maternal trisomic mosaicism, placental trisomic mosaicism.

A sample with a fetal fraction of 10% and a z-score of 15 is provided, which is slightly above the T _o p band of trisomy as indicated by the conventional [-3,3] band of disomy. Such a sample would be considered to show tetrasomic placental mosaicism if the mother were euploid. In a mother with 7% trisomic mosaicism

SK 50048-2021 A3, this sample would show 50% to 100% trisomic placental mosaicism. In other words, trisomic mosaicism could be mistaken for tetrasomic if the mother is considered euploid.

Example 5 (7o.i7b.29): Maternal trisomic mosaicism, placental trisomic mosaicism.

A sample with a 10% fetal fraction and a z-score of 13.2 is provided, and the z-score range for euploid samples is [-3.3]. If the mother is known to show 10% mosaicism on the chromosome, then the trisomic mosaicism of the placentas can be estimated at [0,0.58], i.e. j. the sample is compatible with placental euploidy. However, if the mother were assumed to be euploid, the placenta would be compatible with trisomy, since the degree of trisomic placental mosaicism would be in the interval [0.9,1],

Example 6 (7o.o7Mo,62): Maternal trisomic mosaicism, placental monosomic mosaicism.

A sample with a fetal fraction of 10% and a z-score of 0 is given. Samples with two copies of the chromosome have z-scores in [- 2,7,2,7], as is the case for the X chromosome subset used to determine gender. Then, assuming the mother is euploid (ie has two copies of the chromosome), the sample would also be considered euploid. In a mother with 7% trisomic mosaicism (XXX, for chromosome X), this sample would show 26% to 99% (mean, 62%) monosomic placental mosaicism; thus allowing the male gender of the placenta. At 10% fetal fraction, a girl from a healthy mother is indistinguishable from a boy from a mother with 7% XXX mosaicism.

Example 7 (Τ0.1Λ/0.9): Maternal trisomic mosaicism, placental monosomic mosaicism.

Given a sample with a fetal fraction of 10% and a z-score of -2. Samples with two copies of a chromosome have z-scores in [-3,3], as is the case for (subset of) chromosome 21. Then assuming that the mother is euploid (ie has two copies of a chromosome), the sample would also be considered euploid. For a mother with 10% trisomic mosaicism, this sample would show 80% to 100% (mean, 90%) monosomic placental mosaicism. Thus, at 10% fetal fraction, a euploid placenta from an euploid mother is indistinguishable from a monosomal placenta from a mother with 10% T21 mosaicism.

Example 8 (MoaToas)· Maternal monosomic mosaicism, placentas trisomic mosaicism.

A sample with a fetal fraction of 10% and a z-score of 6.6 is provided. Assume that samples with two copies of the chromosome have z-scores in [- 2.7, 2.7], as is the case for the X chromosome subset used to determine sex. Then, assuming the mother is euploid (i.e. has two copies of the X chromosome), the placenta should be considered male-bearing; see Figure 10. For a mother with 10% monosomic mosaicism (XO, for the X chromosome), this sample would show 0% to 36% (mean, 18%) trisomic placental mosaicism; thus, it allows for a female placenta (on the low end) or 36% XXX placental mosaicism (on the long end). With a 10% fetal fraction, a boy from a healthy mother is indistinguishable from a girl from a mother with a 10% X0 mosaic.

Example 9 (Λ/01Μ0.17): Maternal monosomic mosaicism, placental monosomic mosaicism

The same sample as in the previous example 8 is provided. The sample has a fetal fraction of 10% and a z-score of -6.6 on chromosome X. Such a sample is male (by X chromosome) provided the mother has a healthy karyotype. For a mother with 10% monosomic mosaicism (X0, for the X chromosome), this sample would show 0% to 34% (mean, 17%) monosomic placental mosaicism; therefore, it would be compatible with female gender (at the low end) and with 34% monosomic mosaicism at the other end.

Claims (33)

SK 50048-2021 A3 PATENT CLAIMS 1. Method for determining the uncertainty of the degree of trisomic mosaicism in a sample with maternal trisomic mosaicism of degree μ using the interval [y _m i _n , y _max ] of trisomic placental mosaicism with width w = y _m!lx - 7 _m i _n where while • t is the representation of the chromosome reading of the sample, • /is the fetal fraction of the sample, • is the minimum value of the representation of the chromosome reading in the set of euploid samples, • ^{ŕmax is} the maximum value of the representation of the chromosome reading in the set of euploid samples, and assuming that tmin (1 + 0.5 μ [1 - /]), at — tmax ' (1 3 0·5 ' μ [1 ^— /]) + 0.5 tmax f 2. The method according to claim 1, where the degree of trisomic mosaicism in a sample with maternal trisomic mosaicism of degree μ is determined using the midpoint of the trisomic mosaicism interval of the sample, ie using γ = 0.5 · (y _min + 7 _max ). 3. A method for determining whether a sample characterized by its fetal fraction/chromosome read representation t, maternal trisomic mosaicism grade μ and trisomic mosaicism interval width w as defined in claim 1 is eligible or not eligible for determining the degree of trisomic mosaicism with a maximum uncertainty w', including determining whether w < w', where if w > w', said sample is not suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w ', or where, if w < w'. said sample is capable of determining the degree of trisomic mosaicism with the maximum uncertainty w'. 4. The method of any one of claims 1 to 3, wherein in the sample with maternal trisomic mosaicism, the trisomic mosaicism involves any autosome or sex chromosome. 5. The method according to any one of claims 1 to 4, wherein the representation of the t chromosome read is replaced by z-score z, ί-μ ^Ε Z - σ ^Ε Where μ ^Ε is an estimate of the location of the distribution of chromosome read representation in a population that is euploid in chromosome, </is an estimate of the extent of the distribution of representation of chromosome reads in a population that is euploid in chromosome, including the determination of the uncertainty of the degree of trisomic mosaicism in a sample with maternal trisomic mosaicism of the degree μ using the interval [y _m i _n ,y _max ] of trisomic mosaicism with width w = y _max /min, where /min = max 0.2 z σ ^Ε + μ ^Ε + (σ ^E Zmax + μ ^£ ) (0.5 μ ( / - 1) - 1) /(^zLx + ú*) where z^™ is the minimum z-score in the set of samples that are euploid for the chromosome, and z^ax is the maximum z-score in the set of samples that are euploid for chromosome, and provided that z > z ^E _in (1 + 0.5 μ [1 - /]) + 0.5 _and z < z ^E _ax (1 + 0.5 [μ (1 - /) + /]) + 0.5 . 6. The method according to any one of claims 1 to 5, where μ = 0. 7. The method according to claim 6, wherein said method for determining whether a sample is capable or not capable of determining the degree of trisomic mosaicism with the maximum uncertainty w' is equivalent to determining whether SK 50048-2021 A3 t^tmin + 0.5-w'-/-t“ _in ,(1) t tmax + 0-5 ' (1 - W ^z ) / t ^E _lax ,(2) (t - tmax) ( /ĽoD - /Ľod) ž - t^) (f -(3) where • r is the representation of the chromosome reading of the sample, • /is the fetal fraction of the sample, • t^in is the minimum value of the representation of the chromosome reading in the set of euploid samples, • ^ŕ max Is the maximum value of the chromosome reading representation in the set of euploid samples, at ^E — t ^E „cr _ ^L max ^c min /ĽoD = 2 — t ^E _____'max ' ^{min_____ tmin - (1 - W') ' t E iax} _{. t} E . _r E ^wt max ^L mi /lod t ^E . — tl — wT t ^E ^min > ^{1 w} J ^max where if all points (1) to (3) are met, the said sample is not suitable for determining the grade of trisomic mosaicism with maximum uncertainty w', or where, if any of (1) to (3) is not met, said sample is suitable for determining the degree of trisomic mosaicism with maximum uncertainty w'. 8. Method for determining the uncertainty of the degree of monosomic mosaicism in a sample with maternal m trisomic mosaicism of degree μ using the interval 1/^, p _max ] of monosomic mosaicism with width v = p _max — p _min where t + Pmin = max 0.-2-• (0.5 ·μ-(/-1)-1) /'^in Pmax = min 1, -2 t + tn _lax (0.5 μ -(/-1)-1) ft ^E J 'max while - t is the representation of the chromosome reading of the sample, - /is the fetal fraction of the sample, - t^in is the minimum value of the chromosome reading representation in the set of euploid samples, - ^ŕ max ^is the maximum value of the chromosome reading representation in the set of euploid samples, and assuming that t > tmin (1 + °· ⁵ [ú (1 - /) - /]), 3 t < t^ _ax (1 + 0.5 ·μ [1 —/]). 9. The method according to claim 8, where the degree of monosomic mosaicism in a sample with maternal trisomic mosaicism of the degree is determined using the midpoint of the monosomic interval of the sample, ie using p = 0.5' (/min + Pm _aX )· 10. A method for determining whether a sample characterized by its fetal fraction/representation of chromosome t reads, maternal trisomic mosaicism grade μ and the width of the v monosomic mosaicism interval as defined in claim 8 is capable or not capable of determining the degree of monosomic mosaicism with maximum uncertainty v', comprising determining whether, v < v', where if v > v', said sample is not eligible for determining the degree of monosomic mosaicism with maximum uncertainty v', or where, if v < v', said sample is eligible for determining the degree of monosomic mosaicism with maximum uncertainty in'. 11. The method of any one of claims 8 to 10, wherein the monosomic mosaicism relates to any autosome or sex chromosome. 12. The method according to any one of claims 9 to 11, wherein the chromosome read representation is replaced by z-score z, SK 50048-2021 A3 where μ ^ζ is an estimate of the location of the distribution of chromosome read representation in a population that is euploid in chromosome, is an estimate of the extent of the distribution of representation of chromosome reads in a population that is euploid in chromosome, including the determination of the uncertainty of the degree of monosomic mosaicism in a sample with maternal trisomic mosaicism of degree μ using the interval |p _min , Pmax] monosomic mosaicism with width T Pmax Pmin where Amn = max 0, -2 ζ·σ ^Ε + \v ^E + (a ^E z ^E _in + μ ^Ε ) (0.5 μ (f - 1) - ľ)' /U ^E '4in + ú ^£ ) . Λ ₉ z + μ ^£ + (σ ^Β Zm _ax +p ^E ) (θ·5 μ (/— 1) — 1) \ Pmax = min 1,-2--——g—g—-g------------ , V f Zmax + t ¹ ) / where z^™ is the minimum z-score in the set of samples that are euploid for the chromosome, and az^ _ax is the maximum z-score in the set of samples that are euploid for the chromosome, and provided that z > 4 _in (1 + 0.5 [μ (1 - /] - Γ) + 0.5 z < · (1 + 0.5 μ [1. - /]) + 0.5 13. The method according to any one of claims 8 to 12, wherein μ = 0. 14. The method according to claim 13, wherein said method for determining whether or not a sample is suitable for determining the degree of monosomic mosaicism with the maximum uncertainty v' is equivalent to determining whether t > ŕ/ _n - 0.5 (1 - v') / 4 _in , (4) t < t£ _ax — 0.5 v' ft™ _ax , (t - 4in) ' (/ld - /lod) > (t _/L M _D - 4in) ' (/ - /lod)(5 ) (6) where - í is the representation of the chromosome reading of the sample, - /is the lethal fraction of the sample, - _in is the minimum value of chromosome reading representation in the set of euploid samples, - tmax is the maximum value of chromosome reading representation in the set of euploid samples, at ^E — t ^E · f EM _ n ^niax ^min /l.nD / . f > t ^E — t ^E · c M _ n ____^max ^c min____ /LoD - ^Z ' _t E _ ₍₁ _ _v ^. _t E, > ^L max V- ¹ - J min ^{- 05} ' /lod) ^max where if all points (4) to (6) are met, said sample is not eligible for determining the degree of monosomic mosaicism with the maximum uncertainty v', or where, if any of points (4) to (6) are not met, said sample is eligible to determine the degree of monosomic mosaicism with maximum uncertainty v'. 15. Method for determining the uncertainty of the degree of trisomic mosaicism in a sample with maternal monosomic mosaicism of the μ' degree using the interval [/ _min , / _ma x] of trisomic placental mosaicism with width iv y _ma x — y mi _and where /min = max 0.2 /max = min 1.2 t-tg _lax · (0.5· μ'(/-!) + !) / tmax ŕ-^ _in · (0.5-μ'-(/-1) + 1) / ímin where • t is the representation of the chromosome reading of the sample, • /is the lethal fraction of the sample, • _in is the minimum value of the representation of the chromosome reading in the set of euploid samples, • tmax ^is the maximum value of the representation of the chromosome reading in the set of euploid samples, and assuming that SK 50048-2021 A3 i S tmas. ' (1 - 0.5 0' ~ (1 + μ') /]). 16. Method according to claim 15, where the degree of trisomic mosaicism in a sample with maternal monosomic mosaicism of degree μ' is determined using the midpoint of the trisomic mosaicism interval of the sample, ie, using / = 0.5 · (y' _min + y' _max ). 17. A method for determining whether a sample characterized by its fetal fraction/chromosome read representation t, maternal monosomic mosaicism grade μ' and width of the trisomic mosaic interval w as defined in claim 15 is capable or not capable of determining the degree of trisomic mosaicism with maximum uncertainty w '", including determining whether w" < w'", Where if w > wu the given sample is not suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w'", or where, if w" < w'" the given sample is suitable for determining the degree of trisomic mosaicism with the maximum uncertainty w'". 18. The method of any one of claims 15 to 17, wherein in a sample with maternal monosomic mosaicism, the trisomic mosaicism involves any autosome or sex chromosome. 19. The method according to any one of claims 15 to 18, wherein the representation of the t chromosome read is replaced by the z-score z ťmax = min 1.2 where p ^E ]s is an estimate of the location distribution of the representation of the chromosome read in the population that is euploid in the chromosome, ďjc estimate of the distribution range of the chromosome read representation in a population that is euploid in a chromosome, comprising determining the uncertainty of the degree of trisomic mosaicism in a sample with maternal monosomic mosaicism of degree μ' using the interval [y min, y 'max] of trisomic mosaicism of width w” = y max y min , where , (no ^Z ' ° ^{E +} ' ⁺ ' (°· ⁵ ú' (/ - 1) + 1) v = maxi 0.2 ----------------- -------=-------------------/(σ ^£ ·4ηχ + μ ^Ε ) ζ·σ ^Ε + μ ^Ε -(σ ^Ε ζ ^Ε 1η + μ ^Ε ) · (0.5 μ' (/ - 1) + 1) /(σ ^Ε ·Ζ ^Ε _ίη + μ ^Ε ) where z^ _in is the minimum z-score in the set of samples that are euploid for chromosome, and ^z max is the maximum z-score in the set of samples that are euploid for chromosome a, provided that z > 4 _ic - U - 0.5 - μ ¹ - [1 - /]) - 0.5 - 3 2 5) ' t1 — ú 5 - ]μ· — (1 + μ} - /]) —- 0.5 - -—~ 20. Method for determining the uncertainty of the degree of monosomic mosaicism in a sample with maternal monosomic mosaicism of the degree μ' using the interval [p ' _min , p ' _ma x] of monosomic mosaicism with width v = p ' _max P min, where , fn 9 4 — t^ _ín (0.5 μ'(/ —1) + 1) P min = max (0,-2--—j---------XJ ''min p'max = min 1, -2 tt ^E _ax · (0.5· μ'(/-!) + !) f J ^c max where - í is the representation of the chromosome reading of the sample, - /is the fetal fraction of the sample, ^_ tmin ^is the minimum value of the chromosome reading representation in the set of euploid samples, - ^ŕ max is the maximum value of the chromosome read representation in the set of euploid samples, and provided that t ž ^ŕ min (1 - 0.5 [μ' - (μ' - 1) /]). at <t^ _ax · (1-0.5· μ'[!-/]). SK 50048-2021 A3 21. Method according to claim 8, where the degree of monosomic mosaicism in a sample with maternal monosomic mosaicism of degree μ' is determined using the midpoint of the interval of monosomic mosaicism of the sample, ie, using p' = 0.5 (p' _mjn + p' _max ). 22. A method for determining whether or not a sample characterized by its fetal fraction/read representation of chromosome t, maternal monosomic mosaicism grade μ' and width v' of the monosomic mosaicism interval as defined in claim 20 is capable or not capable of determining the degree of monosomic mosaicism with maximum uncertainty v', comprising determining whether v < v', where if v > v' said sample is not eligible for determining the degree of monosomic mosaicism with the maximum uncertainty of v', or where if v < v' said sample is eligible for determining the degree of monosomic mosaicism with maximum uncertainty v'”. 23. The method of any one of claims 20 to 22, wherein the monosomic mosaicism relates to any autosome or sex chromosome. 24. The method of any one of claims 20 to 23, wherein the read representation of chromosome i is replaced by the z-score zt—μ ^Ε Z = S~' where μ ^Ε is an estimate of the location of the distribution of chromosome read representation in a population that is euploid in chromosome, σ ⁶ is an estimate of the extent of the distribution of representation of chromosome reads in a population that is euploid in chromosome, including the determination of uncertainty in the degree of monosomic mosaicism in the sample with parent monosomic mosaicism of degree μ' using the interval [p' _m \ _a , ρ^χ] of monosomic mosaicism with width V p max p min, where , _ L _Ί z σ ^Ε + μ ^Ε - (σ ^Ε z ^£ _in + μ ^£ ) (0.5 μ' (/ - 1) + l)ý ^Pmin ' /(σ^,, + μ*) / P max z σ ^Ε + μ ^Β - (σ ^Ε z ^E _ax +p ^B ) (0.5 μ' (/ - 1) + 1) Λ(σ ^£ ·4ηχ + μ ^£ ) where ζ^™ is the minimum z-score in the set of samples that are euploid for the chromosome, and ^z max is the maximum z-score in the set of samples that are euploid for the chromosome, and provided , that z > z ^£ _in (l - 0.5 [μ' - (μ' - 1) /]) - 0.5 and <4 _ax (l-0.5· μ'[!-/]) — 0.5· 25. Method for determining the uncertainty of the degree of tetrasomic mosaicism with respect to trisomy in a sample from a euploid mother using the interval of tetrasomic mosaicism [0 _ma x] with width u = 0 _ma x - 0 _m in, where / /f — \\ 0 _min — max ( 0^ 2 · max ( 0, ——I — 1 I, \ \ J ^max // / tt ^E - \ nn · / -i ⁱⁿ min í _d Dmax = 2 · min 1,----f— ^— 1» max if ·/ x / ^e min / where - í is the representation of the chromosome reading of the sample, - /is the fetal fraction of the sample, - _in is the minimum value of the chromosome reading representation in the set of euploid samples, - ŕ^ax ^is the maximum value of the chromosome reading representation in the set of euploid samples, and provided that t > (1 + 0.5 · f) · _in . 26. The method according to claim 25, where the degree of tetrasomic mosaicism with respect to trisomy in a sample from a euploid mother is determined using the midpoint of the tetrasomic mosaicism interval of the sample, ie, using Θ = 0.5 · (0 _min + θ^). 27. A method for determining whether or not a sample from a euploid mother characterized by its fetal fraction/chromosome read representation t and the width of the u tetrasomic mosaicism interval as defined in claim 25 is capable of determining the degree of tetrasomic mosaicism with a maximum uncertainty u', comprising determining whether u < u', where if u > u' the given sample is not suitable for determining the degree of tetrasomic mosaicism with the maximum uncertainty uor SK 50048-2021 A3 where, if u < u', the given sample is suitable for determining the degree of tetrazonic mosaicism with the maximum uncertainty u'. 28. The method according to claim 27, wherein said method for determining whether a sample from an euploid mother is capable or not capable of determining the degree of tetrasomic mosaicism with maximum uncertainty u' is equivalent to determining whether Í 4in + 0-5 (W + 1) f 4 _in ,(7) t < 4 _ax + 0.5 (2 — u') / 4 _ax ,(8) 0 “ ^- ^ΰέ) ^_ ^ ^Γ οό)> where - í is the representation of the chromosome reading of the sample, - /is the fetal fraction of the sample, - 4 _in is the minimum value of chromosome reading representation in the set of euploid samples, - ^ŕ max ^is the maximum value of the chromosome reading representation in the set of euploid samples, and fE — t ^E fTri _ o _____^max '•mm_____ Λ» ⁰ “ + ^ = ^ + 0-5-^)t ^E — cTetra _______max +mm______ ' ^LoD 4 _in - (2- u')· O' ///“ ⁼ ( ¹ +/lo ^B D “) 4ίηwhere, if all points (7) to (9) are met, the said sample is not suitable for determining the degree of tetrasomic mosaicism with the maximum uncertainty u', or where, if any of the points (7) to (9) are not met, the said sample is capable of determining the degree of tetrasomic mosaicism with the maximum uncertainty u'. 29. The method of any one of claims 25 to 28, wherein the tetrasomic mosaicism involves any autosome or X chromosome. 30. The method of any one of claims 27 to 29, wherein the chromosome read representation í is replaced by the z-score z t-^ ^E z = — σ ^Ε where μ ^Ε is an estimate of the location distribution of the chromosome read representation in a population that is chromosome euploid, + jc estimate the extent of the distribution of chromosome read representation in a population that is euploid in the chromosome, involving the determination of the uncertainty of the degree of tetrasomic mosaicism in a sample from a euploid mother using the interval l/min, /mj of tetrasomic mosaicism with width u = 0 _max where (/ z — Z ^E \| 0.2 max I 0,---------- ^max _E I — 1 I, \ f ^max + ^)/ \ -* x 11 L CZ Λ rt x // where z^™ is the minimum z-score in the set of samples that are euploid for the chromosome, and ^z max ^is the maximum z-score in the set of samples that are euploid for the chromosome, "E and provided that z > (1 + 0.5 /) + 0.5 / ^. 31. The method according to claim 3, 10, 17, 22 or 27, where the sample is not suitable for determining the degree of monosomic, trisomic, and tetrasomic mosaicism, if at least one of the widths of the intervals of monosomic, trisomic, or tetrasomic mosaicism is greater than or equal to the corresponding maximum tolerable the width of the respective interval of mosaicism. 32. The method according to any one of the preceding claims, wherein the monosomic, trisomic, or tetrasomic mosaicism is a 100% degree of monosomy, trisomy, or tetrasomy, respectively. SK 50048-2021 A3 33. A method for calibrating a non-invasive prenatal screening (NIPS) system, comprising (a) generating a monosomy, disomy, trisomy, and tetrasomy band based on processed sequence data obtained from a plurality of blood samples from a pregnant subject containing circulating fetal DNA, wherein the fetal karyotype of each sample is known to be euploid and - is the minimum value of chromosome representation in the set of euploid samples, - ^ŕ max ^is the maximum value of the chromosome representation in the set of euploid samples, where (i) the monosomy band is the following: all pairs (f, t) of the fetal fraction/a representation of the reading of the chromosome t such that t > (1 — 0.5 /) t® _in t < (1 - 0.5 f) ' tmax (ii) the disomy band is as follows: all pairs (f, t) of the fetal fraction/a representation of the reading of chromosome t such that r > . ^c — ⁱⁿ nun t < ^v — ^l max (iii) the trisomy band is as follows: all pairs (f, t) of the fetal fraction/a reading representation of chromosome t such that t > (1 + 0.5 /) t£ _in t < (1 + 0.5 /) t ^E _ax (iv) the tetrasomy band is as follows: all pairs (/ t) of the fetal fraction/a representation of the reading of chromosome t such that t > (1 + /) 4 _in t < (1 + f) t® _ax (b) determining whether on based on processed sequence data obtained from a plurality of blood samples from a pregnant subject containing circulating fetal DNA, wherein the fetal karyotype corresponding to each of the samples is known, said monosomic karyotype samples do or do not fall within the monosomy band, said trisomic karyotype samples do or do not fall within the trisomy band . , samples from point (b) with tetrasomic k by ariotype they fall into the tetrasomy belt, the NIPS system is well calibrated; whereas, if samples from (b) with a monosomic karyotype fall outside the monosomy band, or some samples from (b) with a trisomic karyotype fall outside the trisomy band, or some of the samples from (b) with a tetrasomic karyotype fall outside the tetrasomy band as generated in step (a), the NIPS system is not well calibrated. 10 drawings