{'fraction_non_alphanumeric': 0.030327278546851244, 'fraction_numerical': 0.019364647515403698, 'mean_word_length': 4.994244604316547, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 3, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 3, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Noninvasive Prenatal Measurement of the Fetal Genome

The vast majority of prenatal genetic testing requires invasive sampling. Since this poses a risk to the fetus, one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to noninvasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered noninvasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA prior to shotgun sequencing. This approach enables noninvasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Noninvasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.


Supplementary Notes
Proof of principle experiment for the determination of the fetal genome: sequencing a synthetic mixture containing DNA from a HapMap mother-daughter duo Genomic DNA extracted from the cell lines GM12892 (mother) and GM12878 (daughter) were mixed with a mass ratio of 7:3 (i.e. daughter's contribution to the mixture (ε) was 30%). The genomes of these two individuals, together with the father of this trio, were phased (~800,000 loci) in our previous DDP study (Fan et. Al, Nature Biotech, Jan 2011). The mixture was fragmented by sonication to a size range <300bp.
DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing, without further PCR amplification. The library was quantified by digital PCR before loading on to the flow cell. The library was shotgun sequenced on one lane of the flow cell on a GAII, achieving a sequencing depth of 0.25x of the haploid genome. Image analysis and base calling were performed using Illumina's data analysis pipeline 1.6. The reads were aligned to the human genome (hg18) using the algorithm ELAND in the Illumina's data analysis pipeline. A list of allele calls at each base position along each chromosome was obtained using Illumina's CASAVA software (version 1.6).
Only alleles called with quality scores > 30 were used.
To define markers specific for each parental haplotype, we used the data from previous DDP experiment. For both parents, DDP phased ~800,000 markers, which is the number of loci tested on the whole-genome genotyping arrays. We also imputed the untyped loci on each of the parental chromosomes based on the experimentally phased markers and the 1000 Genome Project data (pilot phase data of the CEU population) using the software Impute v1 (-haploid option) to increase the number of available markers by several folds.
The schematic in Figure S2 illustrated the method to deduce the inheritance of parental haplotypes from the DNA mixture. The markers that define each of the maternal haplotypes are the alleles at the heterozygous loci, as long as the haplotype blocks are long (>1Mb). The markers that define each of the paternal haplotypes are the alleles that are present in one paternal haplotype but not in the other paternal haplotype and the two maternal haplotypes. In the ideal case when there is no measurement error, only the markers specific to the transmitted paternal haplotypes would be present in maternal plasma. Even if there is measurement error at individual locus, the sum of count of detected alleles on the transmitted paternal haplotype would be in excess compared to that on the untransmitted one. We divided each chromosome into overlapping bins and compared the representation of each parental pair of haplotypes within each bin by counting the number alleles specific to each of the four parental haplotypes. At a sequencing depth of merely 0.25x of the haploid genome and a bin size of 10Mb, the over-represented parental haplotypes could be unambiguously identified ( Figure S3A-B, black line) and the results agree with the true inheritance (identified by DDP in our previous study) ( Figure S3A,B, pink or blue background). The exception was in regions near heterochromatin and telomeres, where available markers and sequencing reads mapping to those areas were sparse, and within ~400-700kb ( Figure S3C) at which recombination of parental chromosomes had occurred. Overall, ~99.6% of the paternal inheritance (as measured by the size of the haploid genome) and ~98.2% of maternal inheritance of the child's genome could be correctly deduced in this mixture.

Supplementary Tables
Supplementary Table 1. Estimated sampling requirement (N) for noninvasively determining the inheritance of maternal haplotypes using the equation outlined in "Additional Methods". N refers to the cumulative sum of the allele count of all usable markers on the transmitted maternal haplotype.

Supplementary Figures
Supplementary Figure 1. Relationship between fetal DNA fraction in maternal plasma and the required sampling depth for deducing fetal inheritance of maternal haplotypes. The measure of sampling depth is the median number of occurrences of markers per bin on the transmitted maternal haplotype. The predicted sampling requirements for a given fetal DNA fraction at different confidence level are plotted as solid lines. The same prediction can be applied to estimate sampling requirement for counting to differentiate fetal heterozygosity from fetal homozygosity at individual locus where the mother is heterozygous (in such case the bin size is 1).  Figure 2. Outline of the strategy for determining the fetal genome noninvasively when genetic materials from both parents are available. Genome-wide, chromosome length haplotypes of the parents are obtained, for instance, using direct deterministic phasing in this study. The four parental haplotypes are differentiated by the alleles specific to each of them. Molecular counting of parental haplotypes is achieved by shotgun sequencing of maternal plasma DNA. The markers that define each of the maternal haplotypes are the alleles at the heterozygous loci, as long as the haplotype blocks are long (>1Mb). The markers that define each of the paternal haplotypes are the alleles that are present in one paternal haplotype but not in the other paternal haplotype and the two maternal haplotypes. The inheritance of parental haplotypes is determined by summing the count of the number of alleles specific to each of the parental haplotypes. Supplementary Figure 3. Determining the child's inheritance of parental haplotypes in a mixture containing maternal (GM12892) and child's (GM12878) genomic DNA. The markers that define each parental haplotype are derived from the ~800,000 experimentally phased loci (by DPP) and additional imputed loci. A (previous page).
Maternal haplotypes. Each dot on the black line corresponds to the relative representation of the two maternal haplotypes evaluated using the markers lying within a 10Mb region centered at the circle. Each point is accompanied by an error bar that corresponds to the 95% confidence interval for each measurement, estimated by simulating the distribution of reads assuming the count of each maternal haplotype was the mean of a Poisson random variable. Relative representation was calculated with a sliding window of 100kb. The true inheritance of maternal haplotypes, as determined by previous whole-genome haplotyping experiments of the trio, are shown as the background (red: transmitted from mother to daughter; gray: untransmitted; white: heterochromatin/centromere). All chromosomes are plotted with the same length. B (previous page). Paternal haplotypes. White crosses represent the paternal alleles on each of the two paternal haplotypes observed in the sequencing data. Each point on the black line corresponds to the relative representation of the two paternal haplotypes evaluated using the markers lying within a 10Mb region centered at the position of the circle. Relative representation was calculated with a sliding window of 100kb. The true inheritance of paternal haplotypes, as determined by previous whole-genome haplotyping experiments of the trio, are shown as the background (blue: transmitted from father to daughter; gray: untransmitted; white: heterochromatin/centromere). All chromosomes are plotted with the same length. C (above). Resolution of measuring cross-over events. For cross-over events on the maternal chromosomes, the distance between each measured cross-over and the corresponding true cross-over is plotted. For cross-over events on the paternal chromosomes, the width of each measured cross-over event is plotted. The cross-over events are sorted by resolution. Supplementary Figure 5. The distance between each true cross-over on the maternal chromosomes and the respective measured cross-over measured at various sequencing depth and bin sizes. See Table S2 for genome coverage for 'final sequencing depth' and 'initial sequencing depth'. The determination of maternal inheritance at the 'final sequencing depth' is shown in Figure 2 in the main text, while that at the 'initial sequencing depth' is shown in Figure S6. The cross-over events are sorted by the distance. The true cross-overs are determined by aligning the homozygous SNPs of the fetus by cord blood genotyping against the two maternal haplotypes defined by the phased maternal heterozygous SNPs and therefore are defined intervals marked by two adjacent heterozygous SNPs. The midpoint of each interval is treated as location of true cross-over. Two events were missed in P1T1 when the bin size was 15Mb.  2' 3' 4' 5' 6' 7' 8' 9' 10' 11' 12' 13' 14' 15' 16' 17' 18' 19' 20' 21' 22' 23' 24' 25' 26' 27' 28' 29' 30' 31' 32' 33' 34' 35' 36' 37' 38' 39' 40' 41' 42'

Absolute)distance)(bp))of)measured)cross5over)) from)center)of)true)cross5over)
Supplementary Figure 6. Determining inheritance of maternal haplotypes with shallow sequencing for A. P1T1 (bin size 15Mb, ~11.6x haploid genome coverage) and B. P1T2 (bin size 7.5Mb, ~4.0x haploid genome coverage). All cross-overs were identified in P1T2, while 2 cross-overs were missed in P1T1. Both of these events were close to the heterochromatin (chr13 and chr21) resulting in switches of small blocks that contain few markers available for counting. Increasing sequencing depth ultimately detected these block switches (Figure 2 in main text).  Figure 7. Distribution of per base coverage at locations at which mother is homozygous. Blue: a subset of loci of which mother is homozygous but the paternally inherited allele is different from the maternal allele (i.e. paternal specific alleles); red: all loci at which mother is homozygous. . Samples were taken from the cell fraction of maternal blood and cord blood. Primer pairs and probe to 2 DiGeorge region were dgs37 and dgs40. E1F2C1 (chromosome 1) served as a marker to a diploid reference. Primer and probe sequences are listed in SI Table 3. This data verified that both the patient and her newborn have a deletion in the region known to be associated with DiGeorge syndrome.
Supplementary Figure 9. Outline of the experimental method to obtain reference fetal and maternal exomes.  Figure 14. A. ROC curves with varying coverage cutoffs from first trimester in positions where mother is heterozygous and a true positive is defined to be a correct fetal homozygous call. At a false positive rate of 1%, a coverage of 100X would yield ~92% sensitivity whereas a coverage of 300X would yield ~98% sensitivity. B. ROC curves from the first trimester showing the performance of various filtering conditions based on close proximity positions with high minor allele fractions. When n+1 nearby (within 40 bp) positions ("n prox") all exceed a minor allele fraction of m% ("m%"), those positions are removed. Rigorous filtering had a substantial effect on reducing the false positive rate. C. Filtering removed less than 5% of all positions depending on the threshold of the minor allele fraction. Typical regions removed had multiple positions with high minor allele fractions.