We recruited a abounding brace accessory an obstetrics dispensary for the prenatal assay of β-thalassemia. The ancestor was a carrier of the -CTTT 4–base brace (bp) abatement of codons 41/42, and the abounding mother was a carrier of the A→G alteration at nucleotide −28 of the HBB gene (9). Claret samples were taken from the ancestor and from the mother afore chorionic beard sampling (CVS) at 12 weeks of gestation. A allocation of the CVS DNA was stored for the study.
Genome-wide single-nucleotide polymorphism (SNP) genotyping for ~900,000 SNPs was performed for DNA extracted from benevolent and affectionate buffy accoutrement samples, and the CVS sample, with the Affymetrix Genome-Wide Animal SNP Array 6.0 arrangement (table S1). The SNPs were classified into altered categories (Fig. 1). We authentic class 1 SNPs as those for which the ancestor and mother were both zygous, but for a altered allele each. Class 2 SNPs were those in which the ancestor and mother were both zygous, but for the aforementioned allele. Class 3 SNPs were those in which the ancestor was heterozygous and the mother was zygous. Class 4 SNPs were those in which the ancestor was zygous and the mother was heterozygous. Class 5 SNPs were those in which both the ancestor and the mother were heterozygous.
Noninvasive fetal genomic assay from affectionate claret DNA. Affectionate SNP combinations can be aggregate into bristles categories. Categories 1, 2, and 3 acquiesce the basal ambit for affectionate claret DNA sequencing to be established, including the allotment advantage of the fetal genome, apportioned absorption of fetal DNA, and sequencing absurdity rate. Class 3 additionally allows the fetal bequest cachet of SNP alleles altered to the ancestor to be studied. Mutations abnormally agitated by the ancestor can be admired as class 3. Class 4 allows the bequest cachet of the affectionate haplotype to be studied. One appliance is the tracking of fetal bequest of a haplotype block aing to a alteration agitated by the mother. Here, noninvasive fetal genomic assay was agitated out for a ancestors ability prenatal assay for β-thalassemia. Asterisk denotes that advice on the affectionate haplotype is adapted for the RHDO analysis. Class 5 SNPs were not analyzed in this study, but ability be advantageous for the prenatal assay of autosomal backward disorders with affiliated parents or abiogenetic diseases with a able architect effect.
We performed PE sequencing, 50 bp for anniversary end, on DNA extracted from affectionate plasma. Reads (3.931 billion), agnate to an boilerplate of 65-fold advantage of a animal genome, were accumbent to the non–repeat-masked advertence animal genome (Hg18 NCBI.36). For anniversary of the 45,392 class 1 SNPs in this family, the fetus was an astrict heterozygote. The fetal SNP allele affiliated from the ancestor should be readily detected as a altered arrangement in affectionate claret and could be acclimated for belief the administration of fetal DNA sequences beyond the genome in affectionate plasma.
Figure 2A shows the cardinal of times the paternally affiliated fetal alleles for the class 1 SNPs were empiric in affectionate claret as the abyss of sequencing increased. With abstracts from 3.931 billion reads, a fetal allele was empiric at atomic already for 93.94% of these SNPs (table S2). These after-effects were connected with Poisson administration predictions d that the accomplished fetal genome was analogously broadcast in affectionate claret (fig. S1).
Sequencing of fetal and absolute DNA in affectionate plasma. (A) Abyss of advantage of fetal-specific SNP alleles adjoin the cardinal of sequenced reads. (B) Sequencing abyss and GC agreeable beyond the accomplished genome. Chromosome ideograms (outer ring) are aggressive pter-qter in a clockwise administration (centromeres are credible in yellow). Added advance (from alfresco to inside): GC agreeable (green; range, 30 to 55%), absolute sequencing abyss (red; range, 40 to 100 reads per SNP), and fetal-specific apprehend sequencing abyss (blue; range, 1 to 8 reads per SNP). (C) Admeasurement administration of fetal DNA (blue curve), absolute DNA (red curve), and mitochondrial DNA (green burst curve). Numbers denote the DNA admeasurement at the peaks. Schematic illustrations of the structural alignment of a nucleosome are credible aloft the graph. From larboard to right, DNA bifold braid anguish about a nucleosomal bulk assemblage with the sites for nuclease break shown; a nucleosome bulk assemblage with ~146 bp of DNA (red tape) anguish about it; and a nucleosomal bulk assemblage with an complete ~20-bp linker sequence.
The apportioned fetal DNA absorption in the affectionate plasma, f, can be afflicted from the sequencing data:f=2pp qwhere p is the cardinal of sequenced reads of the fetal-specific allele (the A allele for the class 1 SNP in Fig. 1) and q is the apprehend adding of the added allele, which is aggregate by the affectionate and fetal genomes (the C allele for the class 1 SNP in Fig. 1). The ethics of f bent for every chromosome were awful connected (Table 1). The abyss of advantage of fetal and affectionate sequences (in 1-Mb windows) beyond the genome is advised in Fig. 2B. It activated with the GC agreeable of anniversary genomic window (fig. S2). The cardinal of fetal sequences as a admeasurement of the absolute sequences in anniversary window was connected with the apportioned fetal DNA absorption bent on a chromosomal akin (Table 1). These abstracts adumbrated that the about admeasurement of fetal and affectionate DNA was abundantly connected beyond the absolute genome. Antecedent abstracts acquire adapted that the GC bent affecting the altitude of absolute DNA in affectionate claret is acceptable to be an analytic antiquity accompanying to the sequencing belvedere acclimated (6, 7, 10, 11), rather than an adumbration of the cogwheel representation of DNA molecules of altered GC content. Our abstracts accordingly advance that the administration of the fetal and affectionate genomes is almost alike in affectionate plasma.
Fractional concentrations of fetal DNA afflicted based on the assay of class 1 SNPs for altered chromosomes.
The admeasurement of anniversary sequenced claret DNA atom can be deduced from the genome coordinates of the ends of the PE reads. The sizes of the fetal and absolute sequences were bent for the accomplished genome (Fig. 2C) and alone for anniversary chromosome (fig. S3). The best abounding absolute sequences (predominantly maternal) were 166 bp in length. The best cogent aberration in the admeasurement administration amid the fetal and the absolute DNA was that fetal DNA credible a abridgement in the 166-bp aiguille (Fig. 2C) and a about bulge of the 143-bp peak. The closing acceptable corresponded to the accent of a ~20-bp linker fragment from a nucleosome to its bulk atom of ~146 bp (12). From ~143 bp and below, the distributions of both fetal and absolute DNA accustomed a 10-bp aeon evocative of nuclease-cleaved nucleosomes (12). These abstracts advance that claret DNA bits are acquired from the enzymatic processing of DNA from apoptotic cells. In contrast, admeasurement assay of reads that mapped to the non–histone-bound mitochondrial genome did not appearance this nucleosomal arrangement (Fig. 2C). These after-effects accommodate a atomic account for the ahead appear admeasurement differences amid fetal and affectionate DNA appliance Y chromosome and alleged polymorphic abiogenetic markers (8, 13, 14), and appearance that such admeasurement differences abide beyond the absolute genome.
After accepting accustomed that the absolute fetal genome was analogously represented in affectionate plasma, we attempted to accrue a genome-wide abiogenetic map of the fetus. Affectionate claret DNA molecules are abbreviate bits and the fetal sequences are in the minority. Here, we acclimated the abiogenetic anatomy of the affectionate genomes as scaffolds for accumulating the fetal abiogenetic map from the affectionate claret DNA sequences. The map resolution depends on the accepted resolution of the affectionate genomes.
First, we acclimated the class 2 SNPs (Fig. 1), in which the ancestor and mother were both zygous for the aforementioned allele, to appraisal the absurdity bulk of claret DNA sequencing. For the 500,457 class 2 SNPs in this family, the fetus would be zygous for the alleles concerned. The sequencing absurdity bulk was bidding as the cardinal of reads with an abrupt allele as a admeasurement of all reads accoutrement the class 2 SNP loci and was 0.303% (99,467/32,828,899). These abrupt alleles were credible in 4.04% of the class 2 SNP loci. Suppose that an allele charge be credible added than already to be scored, alone 0.55% of such SNPs had a apocryphal allele credible in at atomic two reads, connected in a specificity of 99.45%. However, back activated to the apprehension of fetal-specific alleles, the claim of two reads bargain the fetal allele apprehension sensitivity. The paternally affiliated fetal allele was credible at atomic alert in alone 81.06% of class 1 SNPs, area both parents were zygous for altered alleles (table S2).
We deduced the fetal bequest from anniversary ancestor in a stepwise fashion. To actuate the benevolent allele that the fetus had inherited, we advised the 129,835 class 3 SNPs area the ancestor was heterozygous and the mother was zygous for one of the alleles. Anniversary of the two benevolent alleles had a 50% adventitious of actuality affiliated by the fetus.
The paternal-specific allele (as illustrated by the C allele in SNP class 3; Fig. 1) was detected at atomic already amid the sequenced reads accoutrement 63,962 class 3 loci and at atomic alert accoutrement 53,070 loci. The CVS genotype abstracts adumbrated that the fetus affiliated the paternal-specific alleles in 65,018 class 3 SNPs. Such paternally affiliated fetal alleles were empiric at atomic already in 61,049 (that is, 93.90%) and at atomic alert in 52,697 (that is, 81.05%) loci, in acceptable acceding with the class 1 SNP ethics (table S2). If we acquire that the genotyping was perfect, the differences in genotyping and sequencing meant that sequenced paternal-specific alleles empiric already or alert in 2913 and 373 class 3 loci, respectively, were apocryphal positives. Accustomed that the CVS genotyping abstracts adumbrated that the fetus affiliated the aforementioned allele from the ancestor as the zygous affectionate allele in 64,817 loci, the specificities for fetal allele apprehension appliance the one- and two-read belief were 95.51 and 99.42%, appropriately (table S2). These absurdity ante are connected with the class 2 SNP results.
For affectionate inheritance, we analyzed the class 4 SNPs (Fig. 1), area the mother was heterozygous and the ancestor was zygous, and asked whether a slight allelic alterity was present in affectionate plasma. An alterity would announce that the fetus was zygous for one affectionate allele. This assay could, in principle, be agitated out for anniversary SNP with locus-specific approaches such as agenda polymerase alternation acknowledgment (PCR) (15). However, for genome-wide accidental sequencing, the abyss of advantage bare and appropriately the costs would be prohibitive for analytic use. Appliance adjacent SNP alleles on the aforementioned affectionate chromosome as a haplotype, we developed a new access to actuate whether there was a about haplotype dosage (RHDO) alterity in affectionate plasma. Because of meiotic recombination, the final maternally acquired haplotype affiliated by the fetus is a circuitous of the two aboriginal affectionate haplotypes. Appliance RHDO analysis, the aggregate of alleles affiliated by the fetus from its mother can again be deduced as a alternation of bequest blocks. The resolution for audition this depends on the cardinal and administration of abiogenetic markers accepted for the mother’s genome.
In this proof-of-concept study, we deduced the affectionate haplotype advice bare for RHDO assay with genotype advice acquired from microarray assay of the CVS. This precluded the absolute ascertainment of affectionate meiotic recombinations, but we do appearance after in the abstraction that the access can ascertain “artificial” affectionate meiotic recombinations. If RHDO is acclimated clinically, the affectionate haplotype can be deduced after any fetal advice by allegory with genotype advice for added ancestors members.
Figure 3 shows the RHDO process. The two affectionate haplotypes are Hap I and Hap II (Fig. 3A). Hap I is the absolute recombinant affectionate chromosome affiliated by the fetus. We advised if the sequencing abstracts accepted that the fetus had affectionate Hap I. Haplotype advice from the ancestor was not all-important for this analysis.
Relative haplotype dosage (RHDO) analysis. (A) In blazon α SNPs, benevolent alleles are identical to the affectionate alleles on Hap I. In blazon β SNPs, benevolent alleles are identical to the affectionate alleles on Hap II. If the fetus inherits Hap I from the mother, it is zygous for blazon α and heterozygous for blazon β SNPs. (B) For blazon α SNPs, Hap I is overrepresented in affectionate plasma. (C) For blazon β SNPs, there is no cogent aberration amid the accumulative counts for Hap I and Hap II SNPs. Accustomed that the fetus in this case inherits Hap II from the father, the after anticipation arrangement assay (SPRT) deduces the bequest of Hap I from the mother.
The class 4 SNPs were disconnected into two types that adapted altered analysis. We authentic α SNPs as those in which the benevolent alleles were the aforementioned as those on affectionate Hap I (Fig. 3A). Fetal bequest of Hap I acquired an overrepresentation of Hap I, about to Hap II, in affectionate claret (Fig. 3B). If the fetus affiliated Hap II, no overrepresentation would be seen. We authentic blazon β SNPs as those in which the benevolent alleles were the aforementioned as those on affectionate Hap II (Fig. 3A) and fetal bequest of Hap I maintained an according representation of Hap I and Hap II in affectionate claret (Fig. 3C). However, if the fetus affiliated Hap II, Hap II would be overrepresented. A after anticipation arrangement assay (SPRT) determines the statistical acceptation of any allelic alterity credible (16). SPRT allows antecedent testing as abstracts accrue (17, 18). Back the allocation beginning for SPRT is reached, the fetal bequest of a accurate bounded affectionate haplotype would be accustomed (Fig. 4).
SPRT classification. (A and B) SPRT allocation action for RHDO assay of (A) blazon α and (B) blazon β SNPs in a arena aing to the pter of chromosome 1. The allocation action runs in the administration from the telomeric end to the centromere. See additionally Tables 2 and 3.
Data bendability was advised in two ways. Haplotype assignments from alone analyzed blazon α and β SNPs should be the aforementioned and should be absolute of the administration forth the chromosome for which RHDO was used. RHDO analyses of a articulation of blazon α and a articulation of blazon β SNPs aing to the telomeric end of chromosome 1 are credible in Fig. 4 and Tables 2 and 3. Both the blazon α and the blazon β SNP analyses deduced the bequest of Hap I by the fetus. Tables S3 and S4 allegorize the RHDO assay for chromosomes 1 and 22. The chromosome 1 assay proceeded from the telomeric end of the p arm (pter) to the telomeric end of the q arm (qter), and the reverse. The chromosome 22 assay proceeded from the centromere to qter, and the reverse. Haplotype classifications for the RHDO segments analyzed from both admonition were consistent. The pter to qter chromosome 1 RHDO assay adapted an boilerplate of 17 blazon α SNPs and 18 blazon β SNPs to actuate the affectionate haplotype affiliated by the fetus. Three hundred and fourteen blazon α and 267 blazon β RHDO classifications were fabricated for chromosome 1 (table S3).
SPRT allocation action for RHDO assay of blazon α SNPs in a arena aing to the pter of chromosome 1.
SPRT allocation action for RHDO assay of blazon β SNPs in a arena aing to the pter of chromosome 1.
Table 4 summarizes the RHDO abstracts for the accomplished genome. There were 3863 and 3469 classified segments for blazon α and β SNPs, respectively. Because Hap I was the affectionate haplotype anesthetized on to the fetus, any Hap II RHDO segments had been classified incorrectly. If all the RHDO classifications were interpreted anon as the haplotype affiliated by the fetus, there were 25 and 43 amiss RHDO classifications for the blazon α and β SNPs, appropriately (0.6 and 1.2% of these classifications). With the accepted sequencing coverage, the beggarly sizes of blazon α and β allocation segments were 659,000 and 768,000 bp, respectively. The attendance of two meiotic recombinations aural such distances would be absurd (19). Therefore, we proposed to acquire a about-face in haplotype alone back two after RHDO segments of the aforementioned blazon (that is, α or β) showed the aforementioned haplotype classification. Appliance this consecutive-block algorithm, we acquired three and six incorrect classifications for the blazon α and β SNPs, appropriately (0.08 and 0.18% of these classifications) (Table 4). This bulk of resolution is acceptable for best purposes, because meiotic recombinations action about already per chromosome arm per bearing (19).
Accuracy of (A) blazon α and (B) blazon β SNP allocation (from pter to qter).
To authenticate that the RHDO action could potentially ascertain affectionate recombination, we alien two approximate bogus recombinations on chromosome 1 (at positions 163,000,000 and 204,815,000) and one on chromosome 22 (at position 34,835,000) by alteration the fetally acquired affectionate haplotypes at these positions. Figure 5 is a schematic analogy of how the area of recombination is pinpointed by RHDO analysis. RHDO assay was agitated out for a chromosomal arena in both admonition (from the telomeric end to the centromere, and from the centromere to the telomeric end). Back RHDO assay was performed in the aboriginal administration (telomeric end to the centromere, as credible in Fig. 5), the RHDO classifications for a cardinal of segments would announce one accurate haplotype (that is, Hap I) (Fig. 5). Back the RHDO articulation encompassing the recombination armpit was reached, the RHDO allocation would change to the added haplotype (that is, Hap II), as credible in Fig. 5. However, the catastrophe SNP for the RHDO articulation aloof afore (that is, added telomeric to) the change in RHDO allocation was amid afore the absolute recombinant site. RHDO assay was again performed in the about-face administration (that is, centromere to telomeric end). On this occasion, the RHDO segments would initially acknowledge the allocation of Hap II. Back the recombinant RHDO articulation was reached, the allocation would change to Hap I. Again, the catastrophe SNP for the RHDO articulation aloof afore (that is, centromeric to) the change in RHDO allocation was amid afore the absolute recombination site. The genomic locations of the catastrophe SNP in anniversary RHDO articulation afore the change in allocation articular by the advanced and about-face admonition would enclose the absolute recombination site.
Principles of free the recombination armpit appliance RHDO analysis. (A) RHDO assay is agitated out for a chromosomal arena in both directions. Baby arrows, SPRT-classified segments; block arrows, segments classified as accepting one identical affectionate haplotype; dejected block arrows, Hap I; red block arrows, Hap II. (B) SPRT ambit for the articulation central the blooming oval. After changes in the atom of absolute reads contributed by Hap I alleles. For abstracts credibility on the left, the alleles on Hap I are affiliated by the fetus, arch to an overrepresentation of Hap I. Therefore, the atom would access the aerial allocation beginning back abstracts credibility accumulate. However, for SNPs distal to the recombination site, the alleles on Hap II are affiliated by the fetus, arch to an according representation of Hap I and Hap II reads in affectionate plasma. Thus, the atom would access 0.5 back added abstracts credibility accumulate. Back the atom is lower than the lower allocation threshold, an SPRT allocation of Hap II is made. The recombination armpit is amid amid the aftermost SNP of the RHDO segments aloof afore the change in haplotype allocation as articular by the RHDO analyses performed in both directions.
The RHDO analyses for the recombinant chromosomes 1 and 22 (tables S5 and S6) accurately appear changes in haplotype classifications from the RHDO articulation encompassing the alien recombination sites. In the chromosome 1 RHDO analysis, the fetus appeared to acquire affiliated Hap I from the mother from the centromere to SNP rs1489331 amid at 162,956,807 (table S5) but affectionate Hap II distal to this point. This adumbrated that a recombination had occurred. The abounding RHDO assay for chromosome 1 showed two recombinations amid positions 162,956,807 and 163,133,120, and amid 204,791,360 and 204,869,063 (table S5). The RHDO assay for chromosome 22 showed one recombination amid positions 34,636,212 and 34,835,716 (table S6). For both chromosomes, the deduced recombination spots were aing to the ones artificially introduced.
We aing activated the access categorical aloft for the prenatal assay of β-thalassemia, an autosomal backward claret disease. The disease, characterized by astringent anemia, is due to mutations in the HBB gene on chromosome 11 that encodes the β subunit of hemoglobin. An afflicted fetus charge accede aberrant alleles from both parents. Figure 6A illustrates the area of the benevolent and affectionate mutations in the HBB gene. The DNA sequencing abstracts showed 12 reads with the benevolent codon 41/42 alteration (Fig. 6B), advertence that the fetus had affiliated this mutation. One hundred and eighteen wild-type arrangement reads at codons 41/42 (fig. S4) were seen. The credible apportioned fetal DNA absorption was 18%, accordant with class 1 SNP estimates (Table 1).
Prenatal assay of β-thalassemia by sequencing from affectionate plasma. (A) Locations of the affectionate (nucleotide −28 A→G) and benevolent (-CTTT abatement at codons 41/42) mutations. Dejected lines, blocks of blazon α SNPs; blush lines, blocks of blazon β SNPs; blooming boxes, genes in the β-globin cluster. (B) Empiric sequences accustomed the -CTTT deletion. (C) RHDO analysis. Dejected and blush boxes represent SPRT-classified blocks for blazon α (blue) and blazon β (pink) SNPs. Aural anniversary box, the cardinal of SNPs (first row), numbers of cumulated reads for Hap I and Hap II aural the RHDO block (second row), and the allocation aftereffect (third row) are shown. The locations of the boxes accord to the regions credible in (A).
RHDO assay was performed to actuate whether the fetus had affiliated the affectionate nucleotide −28 A→G mutation. In this family, the −28 alteration was on affectionate Hap II; the wild-type allele was on Hap I. Capacity of the blazon α and β RHDO analyses (table S7) both showed fetal bequest of Hap I from the mother (Fig. 6C). This adumbrated that the fetus had affiliated the affectionate wild-type allele. Thus, the fetus was a heterozygous β-thalassemia carrier.
The assay of cell-free fetal DNA in affectionate claret in 1997 has opened up new possibilities for noninvasive prenatal assay (1). However, antecedent assignment in this acreage had about focused on the apprehension of one or a baby cardinal of fetal DNA targets in affectionate claret (5, 20). Afore the present work, it was not bright whether the absolute fetal genome is represented in affectionate plasma, and whether the about accommodation of fetal and affectionate DNA in affectionate claret are constant. For example, Puszyk et al. acclimated real-time PCR to appraise the amounts of a baby cardinal of genomic loci in animal claret and appear that their about amounts were altered (21). In addition example, Fan et al. additionally cautioned that the representation of sequences from altered loci in affectionate claret ability not be according (7). In this report, we acclimated massively alongside sequencing to authenticate that the absolute fetal genome is represented in affectionate plasma, and is present in a connected about admeasurement to affectionate DNA in affectionate claret (Table 1). This advice is important because it demonstrates that a noninvasive genome-wide browse of the fetal genome from affectionate claret is possible. Here, we acclimated genome-wide sequencing of affectionate claret DNA for the prenatal assay of β-thalassemia in a noninvasive way. Thus, a genome-wide browse for diagnosing fetal abiogenetic disorders is additionally possible. With added reductions in the absurdity bulk of massively alongside sequencing platforms, it may be accessible that de novo mutations in the fetal genome could be detected in a cost-effective address by affectionate claret DNA sequencing.
The genome-wide fetal abiogenetic assay declared actuality is abundant added complicated than the use of massively alongside sequencing for the apprehension of fetal chromosomal aneuploidies (6, 7). For aneuploidy detection, one is about alone absorbed in two parameters, namely, the cardinal of arrangement tags and the mapping of these tags to assorted chromosomes. In comparison, to deduce a genome-wide fetal abiogenetic map, one has to assay the sequencing abstracts in the ambience of the affectionate abiogenetic maps and to accrue these abstracts in a alternation of fetal bequest blocks.
The resolution of such fetal abiogenetic assay is bound alone by the abyss of the sequencing and the resolution of the affectionate abiogenetic maps. In this proof-of-concept study, the affectionate haplotype was deduced from the fetal genotype information, which was acquired through assay of a chorionic beard sample. In absolute analytic scenarios, the closing advice would not be available, and the affectionate haplotype could be deduced in a cardinal of ways: (i) by genotype advice from added ancestors members; (ii) by probabilistic answer appliance the accepted haplotype advice aural a population; and (iii) by methods that acquiesce absolute haplotype advice to be generated, for example, by distinct atom assay (22, 23). With the accretion accessibility of alone whole-genome sequencing, it is accessible that the complete genomic sequences of the parents would be available, or could be generated almost inexpensively with third-generation sequencing technologies. In this case, a close-to-complete genomic arrangement of the fetus could be deduced. The contempo advances in breeding haplotype advice from whole-genome arrangement abstracts would facilitate developments in this administration (24).
The RHDO access for elucidating fetal bequest from the mother has cogent advantages over the about alteration dosage (RMD) adjustment that is based on agenda PCR (15). The agenda PCR–based adjustment attempts to accomplish a allocation based on a distinct alteration or polymorphism. It would about crave several thousand-fold advantage of a ambition arena to accomplish this. This would be abstract for appliance on a genome-wide scale, because the costs of bearing such genome-wide advantage would be prohibitive. Conversely, in RHDO analysis, the statistical ability of the adjustment has been added by accumulation the counts from all alleles present in the aforementioned haplotype. As a result, we were able to accomplish a genome-wide assay with 65-fold genomic coverage.
Analysis of the class 5 SNPs (both parents heterozygous) (Fig. 1) was not performed for the accepted abstracts set because of the abridgement of benevolent haplotype information. However, for approaching studies in which both affectionate haplotypes are known, such SNPs would be advantageous for prenatal assay of autosomal backward disorders with affiliated parents or abiogenetic diseases accepting a able architect effect. Because of their abiogenetic relatedness in the ache locus, anniversary ancestor would acquire one archetype of the ache haplotype. RHDO assay in affectionate claret would acknowledge which haplotype(s) the fetus has inherited.
The noninvasive attributes of our access makes it safer than accepted procedures that crave invasive sampling of fetal tissue (for example, amniocentesis and CVS). However, the new access additionally raises a cardinal of ethical, legal, and amusing issues that crave alive altercation amid clinicians, scientists, ethicists, and the community. Examples of such issues accommodate the best adapted way to accommodate abiogenetic counseling for such a almost circuitous test, a consent, the spectrum of fetal abiogenetic characteristics or abnormalities that can be ethically tested, and disinterestedness issues apropos the accouterment of a almost big-ticket test. Such discussions are already beneath way (25–27), but will charge to be expanded.
One administration for approaching development would be to administer the access declared actuality accurately to assorted disease-related genomic regions by targeted sequencing approaches (28, 29). In this way, one could ambition abiogenetic diseases that are accustomed in a accurate population. Such a targeted access would abundantly abate the bulk of sequencing and would acquiesce added samples to be analyzed per sequencing run. Such a development would additionally acquiesce the assay capacity to be counseled accurately for a focused accumulation of disorders.
Our abstraction helps to annotate the assay of claret DNA by absolute the admeasurement aberration amid circulating fetal and absolute DNA with aerial resolution. It is able-bodied accustomed that circulating fetal DNA molecules are about beneath than affectionate DNA in affectionate claret (8). However, the atomic abject of this ascertainment has been unclear. Fan et al. afresh acclimated PE sequencing to abstraction the admeasurement distributions of fetal and absolute DNA in affectionate claret (14). However, because their abstraction alone complex 1 × 107 reads per sample and acclimated a bioinformatics algorithm that analyzed abstracts in 20-bp “bins,” they were alone able to carbon the accustomed ascertainment that fetal DNA was beneath than affectionate DNA in affectionate claret (8), but were not able to access at new mechanistic insights. In our accepted study, we generated 3.931 × 109 reads in the claret abstraction sample and acclimated 1-bp bins in our bioinformatics analysis. As a aftereffect of this college resolution, we were able to beam that the best cogent aberration amid fetal and affectionate DNA in affectionate claret is the abridgement in the 166-bp aiguille about to the 143-bp aiguille (Fig. 2C). The best acceptable account for this aberration is that circulating fetal DNA consists of added molecules in which the ~20-bp linker fragment has been akin from a nucleosome. Because histone H1 binds to the linker fragment, it would be absorbing to analyze whether antibodies targeting H1 ability preferentially bind to the maternally acquired DNA in affectionate plasma, acceptance accessory of circulating fetal DNA by abrogating selection. Furthermore, H1 is accepted to acquire a cardinal of variants, some of them announcement tissue-specific variations in announcement (30). These variants ability be added exploited to differentiate fetal DNA (predominantly placental) from affectionate DNA (predominantly hematopoietic) (31).
Finally, our abstracts potentially acquire implications for added fields involving the apprehension of claret nucleic acids, such as blight assay (32) and the ecology of tissue transplants (33). For example, it would be absorbing to investigate whether key appearance of the high-resolution admeasurement contour for circulating fetal DNA can additionally be credible in circulating bump DNA and donor graft–derived DNA. Variations on the RHDO adjustment ability additionally acquire applications for the apprehension of tumor-associated abiogenetic alterations.
The activity was accustomed by the Joint Chinese University of Hong Kong–Hospital Authority New Territories East Array Analytic Research Ethics Committee. The claret samples were calm from the abounding woman and her bedmate afore CVS with a consent. Peripheral claret samples were centrifuged at 1600g for 10 min at 4°C, and the claret allocation was recentrifuged at 16,000g for 10 min at 4°C (34, 35). The claret corpuscle allocation was recentrifuged at 2500g, and any antithesis claret was removed. DNA from claret (4 ml) and buffy accoutrement was extracted afterward the claret and anatomy aqueous agreement of the QIAamp DSP DNA Claret Mini Kit (Qiagen). The claret DNA was concentrated by a SpeedVac Concentrator (Thermo, SAVANT DNA120) into a final aggregate of 40 μl per case for consecutive DNA sequencing library preparation. DNA was extracted from the CVS sample with the QIAamp Tissue Kit (Qiagen).
The buffy accoutrement DNA of the brace and the CVS DNA were genotyped with the Affymetrix Genome-Wide Animal SNP Array 6.0 arrangement as ahead declared (36). Briefly, genomic DNA was aboriginal digested with the brake agitator Sty I. It was again ligated to a accepted adaptor with T4 DNA ligase. After ligation, the arrangement was PCR-amplified. The articles were subjected to addition annular of assimilation with Nsp I and linker PCR. The PCR articles were again biotin-labeled and alloyed to arrays, which were again fluorescently labeled and scanned to crop a altitude of admixture acuteness for anniversary delving with the GeneChip Scanner 3000 7G (Affymetrix).
The Affymetrix GeneChip Genome-Wide SNP 6.0 arrays were acclimated in affiliation with Affymetrix Genotyping Console adaptation 2.1. For the paternal, maternal, and CVS samples, 99.1, 94.5, and 98.6%, respectively, of the affection ascendancy probes anesthetized the absence affection ascendancy parameters. The microarray arresting for the 906,600 SNP loci for anniversary of the three samples was again analyzed with the Birdseed v2.0 algorithm (37). The Birdseed alarm bulk was >99% for anniversary sample. SNPs (99.09%, 898,365/906,600) were auspiciously alleged for all associates of the leash (table S1A). These SNPs were again subjected to added clarification for abeyant genotyping errors. First, SNPs with biologically absurd genotype combinations amid the leash acquire been removed (details declared in the fable to table S1). Because best of the DNA molecules in affectionate claret are acquired from the mother, we additionally filtered SNPs with affirmation of genotyping errors for the mother as deduced by the sequencing abstracts (details declared in the fable to table S1). Answer of such errors was performed alone for SNPs initially labeled according to the Affymetrix abstracts as categories 1, 3, and 4. A absolute of 0.60% (5407/898,365) of the SNPs initially alleged by Birdseed was filtered. The abstracts appear in this abstraction were acquired from the resultant account of 892,958 SNPs (99.40% of the SNPs auspiciously alleged by Birdseed) (table S1).
Sequencing libraries were complete from the extracted claret DNA with the Paired-End Sequencing Sample Alertness Kit (Illumina) mostly according to the manufacturer’s instructions. Because claret DNA molecules are abbreviate bits by attributes (8), we bare the accomplish of breach and admeasurement another by gel electrophoresis. Briefly, DNA molecules were end-repaired with T4 DNA polymerase and Klenow polymerase, and with T4 polynucleotide kinase to phosphorylate the 5′ ends. A 3′ overhang was created with a 3′-5′ exonuclease–deficient Klenow fragment. Adaptor oligonucleotides were ligated to the adhesive ends. The adaptor-ligated DNA was antiseptic anon with circuit columns of the QIAquick PCR ablution kit (Qiagen) and accomplished by a 15-cycle PCR with Illumina primers.
The DNA library was adulterated so that 36 pM of the DNA library was subjected to admixture assimilate the PE sequencing breeze cells. DNA clusters were generated with an Illumina array abject with Paired-End Array Bearing Kit v2 (Illumina), followed by 51 × 2 cycles of sequencing on a Genome Analyzer IIx (Illumina) with Sequencing Kit v3 (Illumina). Genome Analyzer Sequencing Ascendancy Software (SCS) v2.5, which could accomplish real-time angel assay and abject calling, was acclimated to backpack out the angel processing and abject calling during the allure and imaging cycles of a sequencing run. We acclimated the absence ambit aural the abstracts assay software (SCS v2.5) from Illumina to clarify poor-quality reads. Chastity, which refers to the brightest acuteness acquired over the sum of the brightest and second-brightest intensities, was afflicted for anniversary base/cycle. In the absence setting, a apprehend would be removed if a abstemiousness of beneath than 0.6 is empiric on two or added bases amid the aboriginal 25 bases.
The PE sequencing abstracts were analyzed by agency of the Abbreviate Oligonucleotide Alignment Affairs 2 (SOAP2) in the PE approach (38). For anniversary PE read, 50 bp from anniversary end was accumbent to the non–repeat-masked advertence animal genome (Hg18 NCBI.36). For the alignment of anniversary end, up to two nucleotide mismatches were allowed. The genomic coordinates of these abeyant alignments for the two ends were again analyzed to actuate whether any aggregate would acquiesce the two ends to be accumbent to the aforementioned chromosome with the actual orientation, spanning an admit admeasurement no added than 600 bp, and mapping to a distinct area in the advertence animal genome.
We again sorted for bifold reads amid these PE reads. A bifold apprehend was authentic as a PE apprehend area the admit DNA atom showed identical alpha and end locations on the animal genome. We removed all but one of the bifold PE reads because they were acceptable to be generated by the PCR process. As a result, 16.7% of the absolute (maternal additional fetal) reads and 17.4% of the fetal-specific reads were removed as alike reads. For reads that spanned an SNP locus, we added removed reads that appear a biologically absurd allele according to the benevolent and affectionate genotypes bent by Affymetrix microarray genotyping.
RHDO was performed on class 4 SNPs area the mother was heterozygous and the ancestor was zygous (Fig. 3). RHDO assay could alpha from any genomic location. At the starting location, the cardinal of sequenced reads accoutrement the two alleles at the starting SNP locus was aboriginal examined. SPRT (see after area below) (16–18) was performed at the starting SNP locus to actuate whether the arrangement amid the counts for the two alleles was in allelic antithesis or imbalance, or the bulk of abstracts was bereft to ability a statistical cessation at this point. If the abstracts adumbrated allelic antithesis or imbalance, the SNP locus would be denticulate as Hap I or Hap II depending on whether it is a blazon α or β SNP (see below). However, accustomed the abyss of sequencing performed (tens of reads per SNP locus), there would be bereft statistical aplomb for a genotype alarm to be fabricated with abstracts from aloof one SNP. Hence, the abstracts would be accounted “unclassified” by SPRT. The SPRT action would again abide to accrue apprehend counts from the SNP locus aing on the haplotype. The action connected until the accumulative abstracts for SNPs forth an bequest block accomplished acceptable statistical aplomb for Hap I or Hap II to be scored. The RHDO assay a the chromosome 1p telomere illustrates the SPRT action (Fig. 4 and Tables 2 and 3). In short, affirmation for allelic alterity strengthens by accumulating abstracts forth the chromosome from assorted SNPs.
The accepted genomic adjustment of SNPs included in the haplotype to be interrogated was acclimated to move unidirectionally forth the chromosome. Blazon α and β SNPs were analyzed alone (Fig. 3). In blazon α SNPs, the benevolent alleles were the aforementioned as those on affectionate Hap I. Fetal bequest of Hap I acquired an overrepresentation of Hap I, about to Hap II, in affectionate plasma. If the fetus affiliated Hap II, no overrepresentation would be seen. In blazon β SNPs, the benevolent alleles were the aforementioned as those on affectionate Hap II, and fetal bequest of Hap I maintained an according representation of Hap I and Hap II in affectionate plasma. However, if the fetus affiliated Hap II, Hap II would be overrepresented. After a haplotype alarm was fabricated for a articulation of SNPs, the aing SNP of the aforementioned type, α or β, was acclimated to restart the RHDO analysis. The action was again until the absolute chromosome or genomic arena of absorption had been analyzed.
The SPRT-based allocation of RHDO was performed with a affairs accounting in Python (http://www.python.org/). An allowance arrangement of 1200 was acclimated for the adding of the beginning for accepting the absent or the another hypotheses. The absent antecedent for anniversary SPRT assay was the absence of dosage alterity amid the apprehend counts for the two affectionate haplotypes, Hap I and Hap II. For blazon α SNPs, the another antecedent was the overrepresentation of Hap I. For blazon β SNPs, the another antecedent was the underrepresentation of Hap I. The acumen of appliance a aerial allowance arrangement for SPRT allocation was to abbreviate the adventitious of incorrect allocation because the ample cardinal of SPRT classifications to be fabricated for the accomplished genome. The adding of the aerial and lower boundaries of the SPRT curves was as ahead declared (16, 39). The equations for artful the aerial and lower boundaries of the SPRT curves are as follows:upper boundary=(ln 1200)/N−ln dln gandlower boundary=(ln 1/1200)/N−ln dln gwhere d=1−q11−q0andwhere g=q1(1−q0)q0(1−q1)q0 is the admeasurement of the absolute counts contributed by apprehend counts from Hap I if the fetus had affiliated Hap II from the mother. q1 is the admeasurement of the absolute counts contributed by apprehend counts from Hap I if the fetus had affiliated Hap I from the mother. N is the absolute cardinal of apprehend counts for the classified segment. ln denotes the accustomed logarithm (that is, loge).
For blazon α SNPs, the fetus would be heterozygous if it had affiliated Hap II from the mother. Hence, q0 was according to 0.5. Alternatively, if the fetus had affiliated Hap I from the mother, it would be zygous for the allele affiliated from the father. Because the mother was heterozygous for the SNPs acclimated for the RHDO analysis, the affectionate allele the fetus had affiliated would be overrepresented in affectionate plasma. The accepted bulk of overrepresentation was bent by the apportioned absorption of fetal DNA in the plasma. In this case, the fetal DNA absorption was 11.43%. Therefore, the amount of q1 would be 0.5572 (= 0.5 0.1143/2).
For blazon β SNPs, the fetus would be heterozygous if it had affiliated Hap I from the mother. Hence, q1 was according to 0.5. If the fetus had affiliated Hap II from the mother, the alleles on Hap I would be underrepresented and the bulk of underrepresentation would be abased on the apportioned fetal DNA absorption in affectionate plasma. Therefore, the amount of q0 would be 0.4429 (= 0.5 − 0.1143/2).
Fig. S1. Predicted advantage of fetal alleles adjoin cardinal of sequenced reads produced.
Fig. S2. Correlation amid abyss of advantage and GC agreeable in 1-Mb windows (A) for the accomplished genome and (B) aural alone chromosomes.
Fig. S3. Admeasurement administration of fetal and absolute DNA bits in affectionate claret for alone chromosomes.
Fig. S4. Account of sequenced reads assuming wild-type sequences for codons 41/42 of HBB.
Table S1. The cardinal and admeasurement of SNPs in altered categories for the advised family.
Table S2. Apprehension ante of altered categories of SNPs in affectionate plasma.
Table S3. RHDO assay for chromosome 1.
Table S4. RHDO assay for chromosome 22.
Table S5. RHDO assay for chromosome 1, which includes two bogus recombinations at positions 163,000,000 and 204,815,000.
Table S6. RHDO assay for chromosome 22 including an bogus recombination armpit amid at 34,835,000.
Table S7. RHDO assay for the affectionate alteration in the HBB gene.
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