Method for increasing fetal fraction

Abstract

Provided are: a method for increasing the fetal fraction in a maternal sample by using a size cutoff value of sequence information, a removal rate of sequence information, or a combination thereof; and a computer readable medium having recorded thereon a program applied for the method. Accordingly, prenatal diagnosis results can be obtained with high accuracy, high sensitivity, or high specificity by increasing the fetal fraction in noninvasive prenatal diagnosis.

Description

How to increase fetal fraction

[0001] A method for increasing fetal fraction by exploiting the characteristics of fetal nucleic acid fragment size in silico (computer-generated simulation) is provided.

[0002] Prenatal diagnosis refers to diagnosing the presence or absence of a fetal disease before birth. Prenatal diagnosis can be broadly divided into invasive and noninvasive diagnostic methods. Invasive diagnostic methods include chorionic villus sampling, amniocentesis, and umbilical cordocentesis. Because invasive diagnostic methods can shock the fetus during the test, potentially causing miscarriage, disease, or malformation, noninvasive diagnostic methods are being developed. Noninvasive diagnostic methods are screening tests and cannot definitively diagnose fetal disease. Therefore, invasive diagnostic methods are performed when confirmation of noninvasive diagnostic results is required or when noninvasive diagnostic methods are not feasible.

[0003] Recently, massively parallel sequencing of DNA molecules in maternal plasma has demonstrated the noninvasive detection of chromosomal abnormalities, including fetal copy number mutations. Fetal DNA can be detected in maternal plasma and serum from the seventh week of pregnancy, and the amount of fetal DNA in maternal blood increases with the duration of pregnancy. The relative proportion of fetal DNA in maternal blood is called fetal fraction. Fetal fraction is closely related to the accuracy of prenatal diagnostic tests. If the fetal fraction falls below the reference value (approximately 4%), the accuracy of prenatal diagnostic tests decreases, rendering the results ineffective. In this case, the mother must undergo a blood draw to re-diagnose the prenatal diagnosis. Even with re-drawing, the fetal fraction often does not increase, resulting in a low success rate.

[0004] Therefore, to overcome these limitations, it is necessary to develop a method to increase the fetal fraction by approximately 4% or more, thereby improving the accuracy, sensitivity, or specificity of prenatal diagnosis without requiring maternal re-blood sampling.

[0005] Provides a method for increasing fetal fraction.

[0006] A computer-readable medium recording a program applied to perform the above method is provided.

[0007] One aspect is as a method of increasing the fetal fraction,

[0008] A step of obtaining sequence information (Read) of a nucleic acid fragment obtained from a biological sample of a pregnant woman;

[0009] A step of obtaining second sequence information from the sequence information based on a size cutoff value of the sequence information, a removal ratio of the sequence information, or a combination thereof;

[0010] A step of mapping the obtained second sequence information to a human reference genome to assign the sequence information of the nucleic acid fragment to a chromosome; and

[0011] A method is provided, including a step of calculating a fetal DNA fraction based on sequence information of a nucleic acid fragment designated in the above chromosome.

[0012] The term "Fetal Fraction (FF)" or "fraction of fetal nucleic acids" refers to the amount or percentage (%) of fetal nucleic acids among nucleic acids isolated from a biological sample of a pregnant woman. The fetal fraction may be a concentration, a relative percentage, or an absolute amount of fetal nucleic acids. The fetal nucleic acids may be nucleic acids derived from placental trophoblast cells. Factors affecting the fetal fraction may include maternal weight, low gestational age, blood sampling, number of fetuses, death of one fetus in a twin pregnancy, donor egg pregnancy, maternal chromosomal aneuploidy, mosaicism, etc.

[0013] Methods of increasing fetal fraction encompass methods of measuring fetal fraction.

[0014] The method comprises the step of obtaining sequence information (Read) of a nucleic acid fragment obtained from a biological sample of a pregnant woman.

[0015] The woman who is pregnant with the above fetus may be pregnant with a single fetus or twins.

[0016] The biological sample may be blood, plasma, serum, urine, saliva, mucosal secretions, sputum, stool, tears, or a combination thereof. The biological sample may be, for example, peripheral blood plasma. The biological sample may contain fetal nucleic acid. The fetal nucleic acid may be cell-free DNA (cfDNA). The fetal nucleic acid may be an isolated DNA fragment.

[0017] The sequence information (Read) of the above nucleic acid fragment may be sequence information obtained from multiple nucleic acid fragments.

[0018] The step of obtaining sequence information of a nucleic acid fragment obtained from a biological sample of the pregnant woman may include a step of isolating a nucleic acid from the biological sample.

[0019] The method for isolating nucleic acids from the biological sample can be performed by a method known to those skilled in the art. The length of the isolated nucleic acid fragment may be from about 10 bp (base pairs) to about 2000 bp, from about 15 bp to about 1500 bp, from about 20 bp to about 1000 bp, from about 20 bp to about 500 bp, from about 20 bp to about 200 bp, or from about 20 bp to about 100 bp.

[0020] The step of obtaining sequence information of a plurality of nucleic acid fragments from a biological sample obtained from the pregnant woman may include a step of performing massive parallel sequencing of the isolated nucleic acids.

[0021] The term "massive parallel sequencing" may be used interchangeably with next-generation sequencing (NGS) or second-generation sequencing. Massive parallel sequencing refers to techniques that simultaneously sequence millions of nucleic acid fragments. Massive parallel sequencing can be performed in parallel using, for example, the 454 platform (Roche), the GS FLX Titanium, the Illumina MiSeq, the Illumina HiSeq, the Illumina Genome Analyzer, the Solexa platform, the SOLiD System (Applied Biosystems), the Ion Proton (Life Technologies), Complete Genomics, the Helicos Biosciences Heliscope, Pacific Biosciences' Single Molecule Real-Time (SMRT™) technology, or a combination thereof.

[0022] The method may further comprise a step of preparing a nucleic acid library for performing massive parallel sequencing.

[0023] The above nucleic acid library can be prepared according to a massively parallel sequencing method. The nucleic acid library can be prepared according to the instructions of a manufacturer providing massively parallel sequencing.

[0024] The sequence information of the obtained nucleic acid fragment may also be referred to as a read. The sequence information of the obtained nucleic acid fragment may have a certain range of sequencing coverage. The sequencing coverage may also be referred to as sequencing depth. The sequencing coverage may be the average number of times sequence information is sequenced in a sequence reconstructed through massive parallel sequencing. The sequencing coverage may be calculated by the mathematical formula of (length of sequence information x number of sequence information) / haploid genome length. The sequencing coverage can be from about 0.00001 to about 1, from about 0.0001 to about 0.5, from about 0.001 to about 0.1, from about 0.001 to about 0.05, from about 0.003 to about 0.05, from about 0.005 to about 0.05, from about 0.007 to about 0.04, from about 0.01 to about 0.03, from about 0.015 to about 0.03, from about 0.02 to about 0.03, from about 0.025 to about 0.03, from about 0.01 to about 0.025, from about 0.01 to about 0.02, or from about 0.01 to about 0.015.

[0025] The method includes a step of obtaining second sequence information from the sequence information based on a size cutoff value of the sequence information, a removal ratio of the sequence information, or a combination thereof.

[0026] The above two sequence information may be newly obtained sequence information by processing the sequence information of nuclear fragments obtained from a biological sample of the mother. The processing standard may be a size cutoff value of the sequence information, a removal ratio of the sequence information, or a combination thereof.

[0027] The size of the above sequence information (Read Size) is a value indicating the size of the nucleic acid fragment (bp).

[0028] The sequence information of the nucleic acid fragments obtained above can be sorted in order of size. The sequence information of the nucleic acid fragments sorted in order of size can be selected based on a size cutoff value. The cutoff value may also be referred to as a reference value.

[0029] The size cutoff value of the above sequence information may be about 200 bp or less, about 190 bp or less, about 180 bp or less, about 170 bp or less, about 160 bp or less, about 150 bp or less, or about 140 bp or less. The size cutoff value of the above sequence information may be about 160 bp or less to about 140 bp or less, about 155 bp or less to about 140 bp or less, about 150 bp or less to about 140 bp or less, about 145 bp or less to about 140 bp or less, or about 140 bp or less.

[0030] The above sequence information removal ratio may be a ratio for removing sequence information having a size larger than the size cutoff value of the above sequence information.

[0031] Removing the above sequence information may be done randomly.

[0032] The sequence information removal ratio is about 0% to about 100%, about 5% to about 100%, about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to About 100%, about 95% to about 100%, about 0% to about 95%, about 0% to about 90%, about 0% to about 85%, about 0% to about 80%, about 0% to about 75%, about 0% to about 70%, about 0% to about 65%, about 0% to about 60%, about 0% to about 55%, about 0% to about 50%, about 0% to about 45%, about 0% to about 40%, about 0% to about 35%, about 0% to about 30%, about 0% to about 25%, about 0% to about 20%, about 0% to about 15%, about 0% to about 10%, or about 0% to about 5%. The above sequence information removal rate may be about 40% to about 70%.

[0033] The above fetal fraction can be measured by a method selected from the group consisting of a Y chromosome-based method, a SNP-based method, and a machine learning-based method.

[0034] The method includes a step of mapping the obtained second sequence information to a human reference genome to assign the sequence information of the nucleic acid fragment to a chromosome.

[0035] The above chromosome is a thick thread-like or rod-shaped structure that appears in the nucleus when a cell divides, and refers to a structure in which the DNA and histone proteins of a living organism are condensed. The above chromosome refers to both a loose structure and a condensed structure. In the case of humans, they are diploid, and have 22 autosomes, 2 of which exist as homologous chromosomes, and sex chromosomes of 2 X chromosomes (female) or 1 X chromosome and 1 Y chromosome (male). The chromosomes may be selected from the group consisting of chromosomes 1 to 22, the X chromosome, and the Y chromosome.

[0036] The human reference genome may be hg18 or hg19. Sequence information that maps to only one genomic location in the human reference genome can be designated as unique sequence information. The sequence information of a nucleic acid fragment can be designated to a chromosomal location based on the designated unique sequence number. The chromosomal location may be a continuous range on a chromosome having a length of about 5 kb, about 10 kb, about 20 kb, about 50 kb, about 100 kb, about 1000 kb, or more than 2000 kb. The chromosomal location may be a single chromosome.

[0037] After the step of assigning the second sequence information to the chromosome, the method may further include a step of checking the thickness distribution of the second sequence information of the nucleic acid fragment assigned to the chromosome by section and excluding sections with low reliability of the sequence information from the analysis target. The section may be a section set in units of about 5 kb to about 50 kb. For example, the section may be a section set in units of about 10 kb to about 40 kb, about 15 kb to about 30 kb, or about 20 kb to about 25 kb. By setting the section, filtering can be performed using the GC content of the base sequence. In addition, by setting the section, a group of the depth and GC content of the sequence information of the nucleic acid fragment assigned to the chromosome can be formed, and statistical analysis can be possible. Here, the term "GC content" refers to the ratio (%) of guanine (G) and cytosine (C) among the bases that make up DNA. The above GC content can be calculated from the formula GC content = (G+C) / (A+T+G+C).

[0038] The step of excluding a section with low reliability of the above sequence information from the analysis target may include removing a mismatch portion, removing sequence information assigned to multiple sites, removing redundant sequence information, or a combination thereof. To exclude a section with low reliability of the sequence information from the analysis target, quality filtering, trimming, perfect match, removal of sequences assigned to multiple sites, removal of PCR duplicated reads, or a combination thereof may be performed. The quality filtering is a process of extracting sequence information with high quality for the quality of each base sequence obtained during the sequencing process. The trimming is a process of removing a section with poor quality because the quality of the latter part of the base sequence is low due to the characteristics of the sequencing device. For example, the size of the nucleic acid fragment may be trimmed to be about 50 bp or more, greater than about 50 bp, or greater than about 100 bp. For example, the quality value of the nucleic acid fragment may be 20 or more, 30 or more, 40 or more, or 50 or more. The perfect match selects only the base sequences that perfectly match when mapped to the human reference genome. Since sequences that are assigned to multiple locations are likely to be repetitive sequence regions, sequences that are assigned to multiple locations (multi) can be removed from the obtained sequence information. Removing PCR duplicate sequence information means removing the parts that were more amplified due to errors during the sequencing process. In addition, for statistical analysis, a group with a certain degree of even variation must be selected to obtain meaningful results. The non-thick portion is usually the N-region of the chromosome, so it can be removed from the analysis target.

[0039] The method includes a step of calculating a fetal fraction based on sequence information of a nucleic acid fragment designated in the chromosome.

[0040] The method may further include a step of calculating a unique read count of second sequence information of the nucleic acid fragment based on the sequence information of the nucleic acid fragment designated for the chromosome. The unique read count is the number of unique reads.

[0041] The term "Read Count (RC)" or "read count" is a count of the number of sequence information assigned to a location in a gene.

[0042] The above method can be used for prenatal diagnosis without re-blood collection after calculating the fetal fraction based on the sequence information of the nucleic acid fragment designated in the above chromosome.

[0043] The method may further include a step of performing any one invasive method selected from the group consisting of chorionic villus sampling, amniocentesis, and umbilical cord puncture after the prenatal diagnostic test.

[0044]

[0045] Another aspect provides a computer-readable medium having recorded thereon a program applicable to performing a method according to one aspect. The computer-readable medium encompasses a system including the computer-readable medium.

[0046] The method for increasing fetal fraction can be implemented as a program (or application) comprising an executable algorithm that can be executed on a computer. The program can be provided by being stored on a temporary or non-transitory computer-readable medium.

[0047] A non-transitory readable medium refers to a medium that stores data semi-permanently and can be read by a device, rather than a medium that stores data for a short period of time, such as a register, cache, or memory. For example, the application or program may be provided by being stored in a non-transitory readable medium, such as a CD, DVD, hard disk, Blu-ray disk, USB, memory card, ROM (read-only memory), PROM (programmable read only memory), EPROM (erasable PROM: EPROM), EEPROM (electrically EPROM), or flash memory.

[0048] Temporarily readable media refers to various types of RAM, such as Static RAM (SRAM), Dynamic RAM (DRAM), Synchronous DRAM (SDRAM), Double Data Rate SDRAM (DDR SDRAM), Enhanced SDRAM (ESDRAM), Synclink DRAM (SLDRAM), and Direct Rambus RAM (DRRAM).

[0049] According to a method for increasing the fetal fraction of a single-cell embryo, and a computer-readable medium recording a program applied to perform the same, the method can increase the fetal fraction in noninvasive prenatal diagnosis to obtain prenatal diagnosis results with high accuracy, sensitivity, or specificity.

[0050] Figure 1 is a graph showing the frequency (fraction = frequency per unit / total frequency) for read size (bp) of two samples with fetal fractions of 5.042% and 9.498%, respectively.

[0051] Figure 2a is a graph showing the fetal fraction (%) against read size (bp) for samples with fetal fractions (Y chromosome-based) of 5.042% and 9.498%, and Figure 2b is a graph showing the number of unique reads (numbers) against read size (bp).

[0052] Figure 3 is a graph showing the frequency distribution for read sizes when the cutoff values ​​for read sizes are set to 170 bp or less and 140 bp or less for samples with fetal fractions (Y chromosome-based) of 5.042% and 9.498%, respectively.

[0053] Figure 4a is a graph showing the fetal fraction (%) against the read removal rate (%) when the cutoff value of the read size is set to 140 bp or less and reads larger than 140 bp are removed for samples with fetal fractions (Y chromosome-based) of 5.042% and 9.498%, and Figure 4b is a graph showing the number of unique reads (numbers) against the read removal rate (%).

[0054] Figure 5 is a graph showing the frequency according to read size when the cutoff value of read size is set to 140 bp or less and reads larger than 140 bp are removed for samples with fetal fractions (Y chromosome-based) of 5.042% and 9.498%, and when the read removal rate is 50% and 90%.

[0055] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0056] Example 1. Enhancement of fetal fraction

[0057] 1. Preparation of cell-free DNA and DNA libraries for DNA sequencing

[0058] Blood samples from pregnant women representing a total of 15,999 negative samples were collected. Approximately 10 ml of peripheral blood was collected from the subjects and collected in BCT™ tubes (Streck, Omaha, NE, USA). The collected blood samples were centrifuged at 1,200 × g for 15 minutes at 4°C. Plasma was collected and centrifuged again at 16,000 × g for 10 minutes at 4°C. Cell-free DNA (cfDNA) was obtained from the centrifuged plasma using the MagListo™ cfDNA extraction kit (Bioneer, Korea).

[0059] The obtained cfDNA fragments were end-repaired using T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase, and then Agencourt AMPure XP (Fisher Scientific) was used to obtain cfDNA fragments.

[0060] A DNA library for the ion-proton sequencing system was constructed from prepared cfDNA according to the protocol provided by the manufacturer (ThermoFisher Scientific). A 540 chip was used, yielding an average sequencing coverage depth of 0.3x per nucleotide.

[0061] 2. Massive parallel sequencing

[0062] 1. The DNA library prepared as described in was subjected to massive parallel sequencing using the Ion Torrent S5XL™ system (ThermoFisher Scientific).

[0063] Different raw reads were obtained using Ion Torrent Suite™ software (ThermoFisher Scientific). Filtered reads were aligned to the human genome reference sequence hg19 by Burrows-Wheeler transform (BWT). Sequence reads that mapped to only one genomic location in hg19 were designated as unique reads. Approximately 3.3 x10 of the total reads 6 Dog was the unique lead, and the GC content of the total 15,999 samples ranged from approximately 39% to 45%.

[0064] Among the 15,999 samples described in 1, two male fetal samples were randomly selected, each having approximately 10 million unique reads, a GC content of 39.5% or more and less than 42%, and a Y chromosome-based fetal fraction of approximately 5.042% and approximately 9.498%.

[0065] 3. Methods for enhancing fetal fraction

[0066] The size of fetal cell-free DNA fragments is known to be shorter than maternal cell-free DNA fragments. Based on this fact, the following two methods were used to increase the fetal fraction. In the following, the fetal fraction was measured using the Y chromosome-based fetal fraction (Hudecova I et al., (2014) PLoS ONE 9(2): e88484).

[0067] 2. Two methods were performed on two samples randomly selected from the sample with fetal fractions of approximately 5.042% and approximately 9.498%, respectively.

[0068] (1) Method 1

[0069] The frequency of fetal fraction was measured according to read size (bp) in two samples with fetal fractions of approximately 5.042% and approximately 9.498%, respectively, and the results are shown in Figure 1 (x-axis: read size (bp), y-axis: frequency (frequency per unit / total frequency = fraction)). Here, the frequency was calculated including both maternal and fetal cell-free DNA fragments (reads).

[0070] As shown in Fig. 1, the sizes of the entire maternal and fetal cell-free DNA fragments coexisting in the maternal plasma ranged from 20 bp to 350 bp, with the maximum frequency at 170 bp. Since the size of the fetal DNA fragments is relatively shorter than that of the maternal DNA fragments, selecting reads consisting of 140 to 200 bp or less may relatively increase the proportion of fetal cell-free DNA fragments. In addition, samples with a large fetal fraction were distributed more frequently in the region of relatively small read sizes.

[0071] To determine the proportion of fetal cell-free DNA fragments in reads with a read size of 140 bp to 200 bp, the cutoffs of the reads of the above samples were selected as sizes of 200 bp or less, 190 bp or less, 180 bp or less, 170 bp or less, 160 bp or less, 150 bp or less, and 140 bp or less. The results of calculating the fetal fraction (%) for the read size are shown in Fig. 2a and Table 1, and the results of calculating the number of unique reads for the read size are shown in Fig. 2b and Table 2.

[0072] Total Fetal Fraction 5.042% 9.498% Read Size (bp) Fetal Fraction (%) Total Reads 5.042 9.498 200 or less 5.044 9.81 4190 or less 5.094 10.075 180 or less 5.197 10.54 4170 or less 5.756 11.93 5160 or less 7.68 214.95 4150 or less 9.77 317.18 2140 or less 11.24 18.599

[0073] Total Fetal Fraction 5.042% 9.498% Read Size (bp) Unique Read Count Total Reads 10,153,9498,595,384200 or less 9,930,1538,047,391190 or less 9,756,4787,678,547180 or less 9,261,6346,910,450170 or less 7,596,8385,374,286160 or less 4,528,6223,507,432150 or less 2,865,5552,454,264140 or less 1,845,7421,702,699

[0074] As shown in Fig. 2a and Table 1, when the read size was set to a cutoff value of a certain value and reads below the cutoff value were removed, the fetal fraction increased as the read size decreased within the range of 140 bp to 200 bp. In particular, when the cutoff value of the read size was set to 140 bp or less, the fetal fraction increased by about 2 times compared to the total reads. As shown in Fig. 2b and Table 2, as the read size decreased within the range of 140 bp to 200 bp, the number of unique reads decreased.

[0075] For samples with fetal fractions of 5.042% and 9.498%, when the cutoff values ​​for read sizes were selected as 170 bp or less and 140 bp or less, respectively, the frequency distribution according to read size was calculated. The calculated results are shown in Fig. 3. As shown in Fig. 3, when the read size was 140 bp or less, the frequency distribution was more skewed to the left than when the read size was 170 bp or less. In general, the size of fetal DNA fragments tends to be smaller than that of maternal DNA fragments. Therefore, as DNA fragments with smaller sizes were selected, the frequency distribution became more skewed to the left and the fetal fraction also increased.

[0076] (2) Method 2

[0077] Reads with a read size of 140 bp or less were selected, and reads larger than 140 bp were randomly removed with probabilities of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. Because there was significant variation between experiments, each experiment was performed 10 times, and the fetal fraction and number of unique reads were measured as the average values.

[0078] The results of calculating the fetal fraction (%) for the lead removal rate (%) are shown in Fig. 4a and Table 3, and the results of calculating the number of unique leads for the lead removal rate (%) are shown in Fig. 4b and Table 4.

[0079] Total Fetal Fraction 5.042% 9.498% Lead Removal Rate (%) Fetal Fraction (%) 05.04 29.498 105.14 39.699 205.24 49.927 305.399 10.18 4405.598 10.51 1505.81 410.940 606.126 11.47 1706.53 212.23 9807.194 13.35 2908.52 15.17 3100 11.24 18.599

[0080] Total Fetal Fraction 5.042% 9.498% Lead Removal Rate (%) Unique Lead Number 010,153,9498,595,384109,472,0867,978,302208,763,9287,346,914308,027,6816,700,945407,261,0166,039,144506,460,8545,361,403605,625,1844,666,822704,749,7483,954,541803,831,5733,223,732902,865,6112,473,4891001,845,7421,702,699

[0081] As shown in Fig. 4a and Table 3, when reads larger than the cutoff value of 140 bp in read size were removed with a probability of 10% to 100%, the proportion of reads smaller than 140 bp among the total reads increased, and the fetal fraction increased. As shown in Fig. 4b and Table 4, when reads larger than the cutoff value of 140 bp in read size were removed with a probability of 10% to 100%, the proportion of reads smaller than 140 bp among the total reads increased, and the number of unique reads decreased.

[0082] As the read removal ratio increased, relatively more fetal DNA fragments were selected, so the frequency distribution was skewed toward smaller read sizes, and it was confirmed that the fetal fraction actually increased.

[0083] For samples with fetal fractions (Y chromosome-based) of 5.042% and 9.498%, the cutoff value for read size was set to 140 bp or less, and reads larger than 140 bp were removed. When the read removal ratio was 50% and 90%, the frequency according to read size was calculated, and the results are shown in Fig. 5. As shown in Fig. 5, when the read removal ratio was increased, relatively more fetal DNA fragments were selected, so the frequency distribution was skewed toward the side with a small read size, and it was confirmed that the fetal fraction also increased further.

Claims

1. As a method for increasing the fetal fraction, A step of obtaining sequence information (Read) of a nucleic acid fragment obtained from a biological sample of a pregnant woman; A step of obtaining second sequence information from the sequence information based on a size cutoff value of the sequence information, a removal ratio of the sequence information, or a combination thereof; A step of mapping the obtained second sequence information to a human reference genome to designate the second sequence information of the nucleic acid fragment to a chromosome; and A method comprising a step of calculating a fetal fraction based on sequence information of a nucleic acid fragment designated in the above chromosome.

2. A method according to claim 1, wherein the size cutoff value of the sequence information is 200 bp or less, 190 bp or less, 180 bp or less, 170 bp or less, 160 bp or less, 150 bp or less, or 140 bp or less.

3. A method according to claim 2, wherein the size cutoff value of the sequence information is 160 bp or less to 140 bp or less.

4. A method according to claim 1, wherein the sequence information removal ratio is a ratio for removing sequence information having a size larger than a size cutoff value of the sequence information.

5. A method according to claim 4, wherein the sequence information is removed randomly.

6. A method according to claim 1 or claim 4, wherein the sequence information removal ratio is 0% to 100%.

7. A method according to claim 6, wherein the sequence information removal ratio is 10% to 100%.

8. A method according to claim 1, wherein the chromosome is selected from the group consisting of chromosomes 1 to 22, the X chromosome, and the Y chromosome.

9. A method according to claim 1, wherein the fetal fraction is measured by a method selected from the group consisting of a Y chromosome-based method, a SNP-based method, and a machine learning-based method.

10. A method according to claim 1, further comprising a step of calculating a unique read count of second sequence information of the nucleic acid fragment based on sequence information of the nucleic acid fragment designated in the chromosome.

11. A computer-readable medium recording a program applicable to performing a method according to any one of claims 1 to 10.

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