CN109411017A - Method for detecting fetal nucleic acid and diagnosing fetal exception - Google Patents

Abstract

The present invention relates generally to the method for detecting fetal nucleic acid and for the method for diagnosing fetal exception.In certain embodiments, the present invention is provided to measure the method that whether there is fetal nucleic acid in maternal sample, the described method includes: obtaining the doubtful maternal sample including fetal nucleic acid, sequencing reaction is implemented to sample with measure in sample at least partly Y chromosome there are situations, so that it is determined that there are fetal nucleic acids in sample.In other embodiments, the method the present invention is provided to quantitative or qualitative analysis to detect the fetal nucleic acid in maternal sample, and it is unrelated with the detection ability of Y chromosome, especially for the sample for including the normal nucleic acid from female child.

Description

Method for detecting fetal nucleic acid and diagnosing fetal exception

The application be the applying date be on 2 9th, 2011, it is entitled " for examining application No. is 201180019992.7 The divisional application of the patent of invention of the method for surveying fetal nucleic acid and diagnosing fetal exception ".

Related application

This PCT application advocates the equity of U.S. Patent Application Serial Number 12/727,824 submitted on March 19th, 2010 and excellent It first weighs, which is the part for the U.S. Patent Application Serial Number 12/709,057 submitted for 19th for 2 months in 2010 Continue, and 12/709,057 be in 2 months 2005 U.S. Patent Application Serial Numbers 11/067 submitted for 25th, 102 part after It is continuous, and 11/067,102 advocates in 2 months 2004 U.S. Patent Application Serial Numbers 60/548 submitted for 27th, 704 priority And equity, the content of above-mentioned each patent application are incorporated herein by reference with it.

Invention field

The present invention relates generally to the method for detecting fetal nucleic acid and for the method for diagnosing fetal exception.

Background

Fetus aneuploidy (such as Down syndrome, edward's syndrome and Pa Ta syndrome (Patau syndrome)) and its Its chromosome aberration influence 9/1000 life birth (Cunningham etc.,Williams Obstetrics, McGraw-Hill, New York, page 942,2002).Usually by through invasive program such as chorionic villus sampling or amniocentesis acquisition The karyotyping of fetal cell diagnose chromosome abnormality.These programs and the potential material risk of both fetus and mother have It closes.Using the Noninvasive screening with maternal serum markers or ultrasound, but its limited reliability (Fan etc., PNAS, 105 (42): 16266-16271, 2008)。

Since finding complete fetal cell in maternal blood, just there is strong interest to go to attempt these cells As the diagnostic window (Fan etc., PNAS, 105 (42): 16266-16271,2008) in fetus genetic.In maternal circulation There are this discovery of the cell-free fetal nucleic acid of certain amount (about 3%- about 6%), cause for various features exploitation based on non- The prenatal genetic of invasive PCR is tested.The problem of these tests is that the measurement of based on PCR loses the sensitivity to specificity, This makes it be difficult to identify specific mutation.Moreover, because the stochastic behaviour of PCR, has ignored point existed in a small amount in sample Fetal nucleic acid in subgroup, such as the sample from maternal tissue or body fluid.In fact, if in several wheels amplification of beginning not Rare nucleic acid is expanded, then the detection of rare event at any time becomes further can not.

In addition, also there is a possibility that such: the fetal nucleic acid of maternal sample is degraded, and can not repair since the nucleic acid is small It is used for PCR amplification.

Need can Noninvasive detection fetal nucleic acid and diagnosing fetal exception method.

Summary

The present invention relates generally to the method for detecting fetal nucleic acid and for the method for diagnosing fetal exception.Method of the invention Using sequencing technologies, especially monomolecular synthesis order-checking (sequencing-by-synthesis) technology, to detect parent group Knit or body fluid in fetal nucleic acid.Method of the invention is highly sensitive, can be used for detecting microcommunity fetal nucleic acid in maternal sample, And it is not usually required to the nucleic acid in amplification sample.

Method of the invention is related to the nucleic acid sequencing and difference parent and fetal nucleic acid obtained from maternal sample.Parent and Difference between fetal nucleic acid identifies fetal nucleic acid, so that abnormal conditions can be measured based on sequence variations.It is described different Single nucleotide polymorphism, variation motif, inversion, missing, addition can be often measured as or any other nucleic acid is reset or exception.

The method of the present invention is also used for by identifying to the unique nucleic acid of fetus, to measure depositing for fetal nucleic acid in maternal sample ?.For example, people can search the sequence of acquisition and the difference of parent canonical sequence；Or it can be related to identify the Y chromosome in sample Substance.Maternal sample can be tissue or body fluid.In specific embodiments, body fluid is maternal blood, Maternal plasma or parent blood Clearly.

The present invention also provides that there are fetal nucleic acids in maternal sample for example, by searching unique sequence or variant to confirm Method.

Sequencing reaction can be any sequencing reaction.In specific embodiments, sequencing reaction is single-molecule sequencing reaction.It is single Molecule sequencing is such as display in the following documents: (U.S. is special for (U.S. Patent number 7,169,560), Lapidus etc. Lapidus Sharp application number 2009/0191565), Quake etc. (U.S. Patent number 6,818,395), Harris (U.S. Patent number 7,282, 337), Quake etc. (U.S. Patent Application No. 2002/0164629) and Braslavsky etc., PNAS (USA), 100: The content of each of 3960-3964 (2003), these bibliography are incorporated herein by reference with it.

In short, in some embodiments, allow single-chain nucleic acid (such as DNA or cDNA) and being connected to flow cell (flow Cell) the oligonucleotide hybridization on surface.Oligonucleotides can be covalently linked to surface or can be used in addition to covalent linkage as this Field various connections known to a person of ordinary skill in the art.In addition, connection can be to be indirectly connected with, for example, via either directly or indirectly It is connected to the polymerase of the present invention on surface.Surface can be plane or other form, and/or can be porous or non-porous, or The surface for any other type being adapted to known to those of ordinary skill in the art.Then under single-molecule resolution, lead to It crosses the imaging of the addition to polymerase-mediated fluorescence-labeled nucleotides or it is detected by other method to implement nucleic acid Sequencing, the addition are included into growth chain surface oligonucleotides.In certain embodiments, core used in sequencing reaction Thuja acid is not chain termination nucleotide.

Because Y chromosome only just exists when fetal nucleic acid comes from male, if Y dyeing is not detected in the sample Body, then the method for the present invention can further comprise implement quantitative determination to the sequence of acquisition detect fetal nucleic acid there are situations. The quantitative determination includes that copy number is analyzed, sparse allele calls (sparse allele calling), targeting to be sequenced again And breakpoint analysis.

The ability of fetal nucleic acid to develop non-invasive diagnostic measurement whether to evaluate fetus in detection maternal sample With exception.Therefore, another aspect of the present invention is provided for measuring whether fetus has abnormal noninvasive method.The present invention Method can be related to obtain the sample including both parent and fetal nucleic acid, implement sequencing reaction to sample to obtain the core in sample The sequence information of the sequence information of acquisition and reference gene group is compared by sour information, thus measure fetus whether have it is different Often, in test sample at least partly Y chromosome there are situations, and if Y chromosome is not detected in the sample, distinguish True negative and false negative.

The importance of diagnostic assay is difference false negative (there are in fact fetal nucleic acid and be not detected) and true negative The measurement ability of (detecting the nucleic acid from healthy fetus).The method of the present invention provides this ability.If in maternal sample Detecting Y chromosome, then the method for the present invention ensures that the measurement can function completely, because Y chromosome is only related to male, And just exist in maternal sample when only there is male fetus nucleic acid in the sample.Certain methods of the invention provide further Quantitative or qualitative analysis distinguish false negative and true negative, and it is unrelated with the detection ability of Y chromosome, especially for including Sample from the normal nucleic acid of female child.The additional quantitative analysis may include copy number analysis, sparse allele tune It is sequenced again and breakpoint analysis with, targeting.

Whether another aspect of the present invention is provided has an abnormal method for measuring fetus, including obtain comprising parent and The maternal sample of both fetal nucleic acids；Unique label is allowed to connect with the nucleic acid in sample, wherein every kind of label and different dyes Colour solid association；Sequencing reaction is implemented to obtain the sequence of tape label to the nucleic acid of tape label；By quantify tape label sequence come Whether measurement fetus has exception.In certain embodiments, label includes unique nucleic acid sequence.

Brief description

Fig. 1 is the histogram for showing the difference between an individual (" itself ") and two family members (" family "), is represented The comparison of three sample rooms, one group of known single nucleotide variant.

Fig. 2 is that display is read derived from single-molecule sequencing and with the HapMap DNA sequence dna uniquely compared referring to human genome Table.Each column represent list HELISCOPE sequenator (single-molecule sequencing instrument, Helicos BioSciences Corporation) the data in channel.

Fig. 3 is the table of the chromosome reading after each sample standard of display.Individual chromosome is counted divided by total Autosome counts.

Fig. 4 is the table for showing the standardised amount of each chromosome.The average mark of reading corresponds in all samples Each chromosome.

Fig. 5 is the diagram of quantitative chromosome counting.

Fig. 6 is the chart for the sample for showing that GC preference (bias) causes chromosome counting to have deflection.

Fig. 7 is genome units (genomic bin) chart that display is drawn as the function of G/C content in unit.? In Fig. 7, sample above is shown to be positively correlated with G/C content, and following sample is shown and G/C content negative correlation.

The A group of Fig. 8 be shown in selected under given G/C content certain genome units for analysis chart.The B group of Fig. 8 Show the sequence information before the correction of GC preference.The C group of Fig. 8 shows the sequence information after the correction of GC preference.

The A group and B group of Fig. 9 shows the sequence information before the correction of GC preference.The C group and D group of Fig. 9 shows the correction of GC preference Sequence information later.

The analysis result of Figure 10 display sequence information.

It is described in detail

For in detection maternal sample fetal nucleic acid there are situation, the method for the present invention uses sequencing reaction.The method of the present invention also makes The maternal blood under genetic condition is analyzed with sequencing reaction, wherein analysis maternal blood in the mixing nucleic acid of fetus and parent with Fetus mutation or genetic abnormality are distinguished from maternal nucleic acids background.

Fetal nucleic acid includes foetal DNA and two kinds of fetal rna.Such as in Ng, mRNA of placental origin is Readily detectable in maternal plasma (can detect placenta source in Maternal plasma easily MRNA), described in (2003) Proc. Nat. Acad. Sci. 100 (8): 4748-4753.

Sample

The method of the present invention is related to obtaining the doubtful sample including both parent and fetal nucleic acid, such as tissue or body fluid.The sample Product may include saliva, urine, tear, vaginal fluid, amniotic fluid, lotion (breast fluid), breast milk, sweat or tissue.? In certain embodiments, which is derived from maternal blood, and in blood plasma, rather than discovery has Circulating DNA in cell.It is preferred that Sample is maternal peripheral venous blood.

In certain embodiments, about 10-20 mL blood is extracted.The blood of the quantity can be such that people obtain at least about (estimated value of the sample size based on fetal nucleic acid, the estimated value are in early pregnancy to the total nucleic acid of 10,000 genome equivalents When about 25 genome equivalent/mL Maternal plasmas, and fetal nucleic acid concentration account for about total blood plasma nucleic acid about 3.4%).However, right It in genetic screening, needs lower statistical significance or nucleic acid samples rich in fetal nucleic acid, therefore less blood can be extracted.

It is identical to be obtained from sample because the amount of fetal nucleic acid usually increases with gestation progresses in maternal sample Or the fetal nucleic acid of similar amt, it can be less with sample needed for gestation progresses.

Enrichment

It in certain embodiments, can be optionally by known method come in enriched sample (such as blood, blood plasma or serum) Fetal nucleic acid, such as select with size fractionation the DNA fragmentation less than about 300 bp.Alternatively, greater than about 500 can be excluded to tend to The mother body D NA of bp.

It in certain embodiments, can be as described such as Li et al. (J. Amer. Med. Assoc. 293:843-849,2005) Maternal blood is handled to increase concentration of the foetal DNA in total DNA, document content is with it entirely through being incorporated by this Text.In short, with commercially available column technology (the highly purified template DNA purification kit of Roche；Roche, Basel, Switzerland) combined vacuum pumps, and extracts Circulating DNA from 5 mL-10 mL Maternal plasmas.After extraction, Ago-Gel is used (1%) electrophoresis (Invitrogen, Basel, Switzerland) separates DNA, carefully cuts off containing about 300 bp's of size The gel section of Circulating DNA.By with extracts kit (11 gel extraction kit of QIAEX；Qiagen, Basel, Switzerland DNA) is extracted from the gel slice, is eluted to the Tris hydrochloric acid that final volume is the 40 sterile 10-mM of μ L In, pH 8.0 (Roche).

It can be by known method come concentration of DNA, including centrifugation and various enzyme inhibitors.DNA is integrated on selective membrane (such as silica) is with by itself and separated from contaminants.Preferred pin carrys out enrichment DNA to the segment recycled in blood plasma, and length is less than 1000 base-pairs, usually less than 300 bp.It is big that this is carried out on DNA size separation medium such as running gel or chromatographic material Small selection.Huber etc. (Nucleic Acids Res. 21 (5): 1061-1066,1993) elaborates the material, Kato etc. (J. Biochem, 95 (1): 83-86,1984) elaborate gel filtration chromatography, tsk gel.It is each in these bibliography The content of a piece is incorporated herein by reference with it.

In addition, certain allele can be inhibited to complete to be enriched with by using peptide nucleic acid (PNA), the peptide nucleic acid is mutual with it The target sequence of benefit combines, but does not expand.

Enders etc. (Clinical Chemistry 49:727-731,2003) elaborates the extraction of blood plasma RNA, in Rong Yiqi is incorporated herein by reference.As described there, by the blood plasma harvested after centrifugation step and Trizol LS reagent (Invitrogen) it is mixed with chloroform.Mixture is centrifuged, shifts water layer into new pipe.Ethyl alcohol is added in water layer.So Suggest afterwards according to producer, add mixture to the mini column of RNeasy (Qiagen) and handles.

Another enriching step can such as Dhallan (J. Am. Med. Soc. 291 (9): 1114-1119,2004 March in year；With U.S. Patent Application No. 20040137470) it is described, with formaldehyde treated blood sample, the content of each of the above document It is incorporated herein by reference with it.Dhallan etc. (U.S. Patent Application No. 20040137470) elaborates foetal DNA Enrichment procedures, wherein by blood collection into the Vacuette pipe (catalog number (Cat.No.) NC9897284) containing 9 ml EDTA, by 0.225 10% neutral buffered liquid of the ml containing formaldehyde (4% w/v) is added in each pipe, slightly reverses each branch pipe.Pipe is stored in 4 DEG C Until for handling.

The reagent of block cell cracking or stabilizing cell membrane, including but not limited to formaldehyde and formaldehyde-derived can be added into pipe Object, formalin, glutaraldehyde and Glutaraldehyde Derivative, crosslinking agent, primary amine reaction crosslinking agent, the agent of sulfydryl cross-linking reaction, sulfydryl add Add object (sulfhydryl addition) or disulfide reduction object (disulfide reduction), carbohydrate reaction Crosslinking agent, the agent of carboxyl cross-linking reaction, light reaction crosslinking agent, crosslinking agent of cleavable etc..Stabilizing cell membrane can be added or hinder thin The reagent of any concentration of cellular lysate.In certain embodiments, stablized with not hindering or interfering the concentration of subsequent reactions to be added Cell membrane or the reagent of block cell cracking.

Flow cytometry also can be used for enriches fetal cells (Herzenberg etc., PNAS 76:1453-1455,1979； Bianchi etc., PNAS 87:3279-3283,1990；Bruch etc., Prenatal Diagnosis 11:787-798, 1991).Saunders etc. (U.S. Patent number 5,432,054) also elaborate using having of manufacture by polyethylene it is wide push up, it is narrow The pipe at capillary bottom carrys out the technology of isolating fetal erythroblast.Lead to density of the red blood cell based on molecule using ramped procedure centrifugation It is accumulated in capillary.Recycling contains the density components of low-density red blood cell (including fetal red blood cells), then differentiation haemolysis With preferential destruction maternal red blood cells.Carry out separating red corpuscle using the density gradient in hypertonic medium, now from lymphocyte and broken Enriches fetal red blood cell in the mother cell split.The use of hypertonic solution reduces red blood cell, and which increase its density, facilitates It is purified from denser lymphocyte.After isolating fetal cell, foetal DNA can be purified with this field standard technique.

Moreover, the reagent of stabilizing cell membrane can be added in maternal blood to reduce mother cell cracking, the reagent Including but not limited to: aldehyde, ureaformaldehyde, phenolic aldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivative, high concentration Magnesium, vitamin E and vitamin e derivative, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivative, bimoclomol, sugarcane Sugar, astaxanthin, glucose, amitriptyline (amitriptyline), hopance tetraphenyl acetate isomers A (isomer A Hopane tetral phenylacetate), hopance tetraphenyl acetate isomers B, citicoline, inositol, vitamin B, dimension Raw element B compound, cholesterol hemisuccinate, sorbierite, calcium, ubiquinone, ubiquinone, vitamin K, vitamin K compound, first naphthalene Quinone, Zonisamide (zonegran), zinc, ginkgo biloba extract, dilantin, perftoran, polyvinylpyrrolidone, phosphorus Acyl serine, Tegretol (tegretol), PABA, disodium chromoglycate, nedocromil (nedocromil) sodium, phenytoinum naticum (phenyloin), zinc citrate, mexitil (mexitil), Di Lanting (dilantin), Sodium Hyaluronate or poloxamer (polaxamer)188。

Example using the scheme of this reagent is as follows: by blood preseration at 4 DEG C until processing.Zero is set as in braking power Centrifuge in 1000 rpm allow pipe be centrifuged ten minutes.It will be centrifuged ten minutes with 1000 rpm for second of pipe.By every a sample Supernatant (blood plasma) be transferred in new pipe, braking power be set as zero when with 3000 rpm centrifugation ten minutes.Supernatant is shifted Into new pipe, -80 DEG C of preservations.About two milliliters " buffy coat " containing mother cell are placed in independent pipe, be stored in- 80℃。

It can be used for measuring kit (Midi Kit) from the Qiagen for purifying DNA in haemocyte, illustrate according to producer Book (measuring kit, catalog number (Cat.No.) 51183 in QIAmp DNA blood) separates genomic DNA from blood plasma.DNA is eluted to 100 μ l In distilled water.Kit is measured in Qiagen also can be used for the separation of the mother cell contained in " buffy coat " DNA.

It extracts

Nucleic acid is extracted from sample according to means known in the art.See, for example, Maniatis etc., Molecular Cloning: A Laboratory Manual (molecular cloning: experiment guide), Cold Spring Harbor, N.Y., the 280-281 Page, 1982, content is incorporated herein by reference with it.

Measure maternal sample in male fetus nucleic acid there are situations

Then using sequencing reaction analyze the nucleic acid from sample, in test sample at least partly Y chromosome there are situations. For example, Bianchi etc. (PNAS USA, 87:3279-3283,1990) report exists only in 222 bp on Y chromosome galianconism Sequence.(Am J Hum Genet, 62 (4): 768,1998) such as Lo etc. (Lancet, 350:485-487,1997), Lo Report the different Y chromosome sequences for being derived from male fetus respectively with (Clin Chem, 45:1570-1572,1999) such as Smid Column.The content that these articles are each is incorporated herein by reference with it.If detecting that Y is dyed in maternal sample Body, then the method for the present invention ensures in sample to include fetal nucleic acid, because Y chromosome is only related to male, and only in sample There are when male fetus nucleic acid its can just exist in maternal sample.

In certain embodiments, sequencing approach is the single-molecule sequencing by synthetic method.Single-molecule sequencing is for example It is shown in following documents: (the U.S. Patent Application No. 2009/ such as Lapidus etc. (U.S. Patent number 7,169,560), Lapidus 0191565), (beauty such as Quake etc. (U.S. Patent number 6,818,395), Harris (U.S. Patent number 7,282,337), Quake State's number of patent application 2002/0164629) and Braslavsky etc., PNAS (USA), 100:3960-3964 (2003), this The content of each of a little bibliography is incorporated herein by reference with it.

In short, by single-chain nucleic acid (such as DNA or cDNA) and the oligonucleotide hybridization for being connected to flowing pool surface.Few core Thuja acid can be covalently linked to surface or the various companies as known to persons of ordinary skill in the art in addition to covalent linkage can be used It connects.In addition, connection can be to be indirectly connected with, for example, via the polymerase of the present invention for being either directly or indirectly connected to surface.Surface It can be plane or other form, and/or can be to be suitable for connecting known to porous or non-porous or those of ordinary skill in the art The surface of any other type connect.Then pass through the adding to polymerase-mediated fluorescence-labeled nucleotides in single-molecule resolution Addition picture carries out nucleic acid sequencing, and the addition is included into growth chain surface oligonucleotides.In certain embodiments, it surveys Nucleotide used in sequence reaction is not chain termination nucleotide.Following part discusses that the overall of nucleic acid sequencing considers, such as right The useful polymerase of synthesis order-checking, selection, reaction condition, signal detection and the analysis on surface.

Nucleotide

The nucleotide useful to the present invention includes any nucleotide or nucleotide analog, whether naturally occurring or synthesis 's.For example, it is preferable to which nucleotide includes the phosphate of following nucleosides: desoxyadenossine, deoxycytidine, deoxyguanosine, deoxythymidine, gland Glycosides, cytidine, guanosine and uridine.The other nucleotide useful to the present invention include adenine, cytimidine, guanine, thymidine Base, xanthine or hypoxanthine；5-bromouracil, 2-aminopurine, deoxyinosine or methylated cytosine, such as 5- methyl Cytimidine and N4- methoxyl group dideoxycytosine.It also include the base of following polynucleotides analogies: the nucleic acid of such as methylation, Such as 2'-O-methRNA；Peptide nucleic acid；The peptide nucleic acid of modification；Locked nucleic acid with can generally be acted as nucleotide or base With (such as by showing the base complement with one or more base present in DNA or RNA, and/or can base it is mutual Mend property be incorporated to) any other structure division；And including chain termination analog.If it is enjoyed at least one base Base complement, then nucleotide corresponds to specific nucleotide type.

Nucleotide of the present invention for nucleic acid sequencing preferably includes the detectable label that can directly or indirectly detect.It is preferred that marking Note includes optically detectable label, such as fluorescent marker.The example of fluorescent marker includes but is not limited to: Atto dyestuff, 4- second Amide groups -4'- isothiocyanato stilbene -2,2' disulfonic acid；Acridine and derivative: acridine, acridine isothiocyanates；5- (2'- ammonia Ethyl) amino naphthalenes -1- sulfonic acid (EDANS)；Two sulphur of 4- amino-N- [3- vinylsulfonyl) phenyl] naphthalimide -3,5 Acid esters；N- (4- anilino- -1- naphthalene) maleimide；Aminobenzamide；BODIPY；It is bright orange；Cumarin and derivative: fragrant Legumin, 7- amino -4- methylcoumarin (AMC, coumarin 1 20), 7- amino -4- trifluoromethyl cumarin (coumarin 1 51)；Flower Cyanine dyes；Tetrachloro-tetrabromfluorescein (cyanosine)；4', 6- diamidino -2-phenylindone (DAPI)；5'5 "-dibromo-o benzene three Phenol-sulfonephthalein (bromopyrogallol red)；7- lignocaine -3- (4'- Isothiocyanato-phenyl) -4- methylcoumarin；Two sub- second Base pentaacetic acid ester；4,4'- diisothiocyanic acid root closes dihydro-stilbene -2,2'- disulfonic acid；4,4'- diisothiocyanic acid root closes stilbene- 2,2' disulfonic acid；5- [dimethylamino] naphthalene -1- sulfonic acid chloride (DNS, dansyl chloride)；4- dimethylamino phenyl azophenyl -4'- is different Thiocyanates (DABITC)；Yihong and derivative: Yihong, isothiocyanic acid Yihong；Erythrosine and derivative: Erythrosin B, different sulphur cyanogen Sour erythrosine；Ethidium Bromide (ethidium)；Fluorescein and derivative: 5-carboxyfluorescein (FAM), 5- (4,6- dichlorotriazine -2- Base) Aminofluorescein (DTAF), 2', the chloro- 6- Fluoresceincarboxylic acid of 7'- dimethoxy-4 ' ' 5'- bis-, fluorescein, isosulfocyanic acid fluorescence Element, QFITC, (XRITC)；Fluorescamine；IR144；IR1446；Isothiocyanates peacock green；4-methyl umbelliferone o-cresolphthalein；Nitre Base tyrosine；Pararosaniline；It is phenol red；B- phycoerythrin；O-phthalaldehyde；Pyrene and derivative: pyrene, pyrene butyrate (pyrene Butyrate), succinimide 1- pyrene；Butyrate quantum dot (butyrate quantum dot)；Reactive Red 4 (Cibacron.TM. azarin 3B-A)；Rhodamine and derivative: 6- carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), Sulforhodamine B, sulfonic acid chloride rhodamine (Rhod), rhodamine B, Rhodamine 123, rhodamine isothiocyanate X, Sulforhodamine B, sulfonyl chloride derivatives (texas Red), the N of Sulforhodamine 101, Sulforhodamine 101, N, N', N' tetramethyl -6- carboxylic Base rhodamine (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanates (TRITC)；Riboflavin；Rosolic acid；Terbium chela Close derivative；Cy3；Cy5；Cy5.5；Cy7；IRD 700；IRD 800；La Jolta is blue；Phthalocyanine；With naphthalene phthalocyanine.It is preferred that fluorescence Labeled as flower cyanines -3 and Hua Jing -5.The present invention also considers the label of non-fluorescent label, including other optically detectable labels.

Polymerase

Nucleic acid polymerase usually useful to the present invention includes archaeal dna polymerase, RNA polymerase, reverse transcriptase or aforementioned any enzyme Mutation or change form.In addition to other places, also at DNA Replication (DNA replication dna), the second edition, Illustrated in Kornberg and Baker, W. H. Freeman, New York, N.Y. (1991) archaeal dna polymerase and Its characteristic.Known conventional archaeal dna polymerase useful to the present invention includes but is not limited to: fierce hot-bulb bacterium (Pyrococcus furiosus) (Pfu) archaeal dna polymerase (Lundberg etc., 1991, Gene, 108:1, Stratagene), walsh heat Coccus (Pyrococcus woesei) (Pwo) archaeal dna polymerase (Hinnisdaels etc., 1996, Biotechniques, 20:186-8, Boehringer Mannheim), thermus thermophilus (Thermus thermophilus) (Tth) DNA polymerization Enzyme (Myers and Gelfand 1991, Biochemistry 30:7661), bacillus stearothermophilus (Bacillus stearothermophilus) archaeal dna polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32), super good hot archeobacteria (Thermococcus litoralis) (Tli) archaeal dna polymerase (also known as Vent.TM. Archaeal dna polymerase, Cariello etc., 1991, Polynucleotides Res, 19:4193, New England Biolabs), 9 degree of Nm.TM. archaeal dna polymerases (New England Biolabs), Stoffel segments, ThermoSequenase (Amersham Pharmacia Biotech UK), Therminator.TM. (New England Biolabs), sea are dwelt Thermobacillus (Thermotoga maritima) (Tma) archaeal dna polymerase (Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239), thermus aquaticus (Thermus aquaticus) (Taq) archaeal dna polymerase (Chien etc., 1976, J. Bacteoriol, 127:1550), archaeal dna polymerase, thermophilic Archimycetes (Pyrococcus kodakaraensis) KOD DNA Polymerase (Takagi etc., 1997, Appl. Environ. Microbial. 63:4504), JDF-3 archaeal dna polymerase (come Self-heating Coccus (ThermococcusSp.) JDF-3, patent application WO 0132887), hot-bulb Pseudomonas (Pyrococcus) GB-D (PGB-D) archaeal dna polymerase (also known as Deep Vent.TM. archaeal dna polymerase, Juncosa-Ginesta etc., 1994, Biotechniques, 16:820, New England Biolabs), UlTma archaeal dna polymerase (comes adaptive hot sea to dwell heat Robe bacterium (Thermotoga maritima)；Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239；PE Applied Biosystems), Tgo archaeal dna polymerase (from Pyrococcus furiosus (Thermococcus gorgonarius), Roche Molecular Biochemicals), Escherichia coli (E. coli) DNA polymerase i (Lecomte and Doubleday, 1983, Polynucleotides Res. 11:7505), T7 archaeal dna polymerase (Nordstrom etc., 1981, J. Biol. Chem. 256:3112) and archeobacteria (archaeal) DP11/DP2 archaeal dna polymerase 11 (Cann etc., 1998, Proc. Natl. Acad. Sci. USA 95:14250)。

Consider both mesophilic property polymerase or thermophilic polymerase.Thermophilic archaeal dna polymerase includes but is not limited to ThermoSequenase, 9. degree of .Nm.TM., Therminator.TM., Taq, Tne, Tma, Pfu, Tfl, Tth, Tli, Stoffel segment, Vent.TM. and Deep Vent.TM. archaeal dna polymerase, KOD archaeal dna polymerase, Tgo, JDF-3 and its mutation Body, variant and derivative.The highly preferred form of physiologic variables of any polymerase are 3' exonuclease-deficiency mutant.

The reverse transcriptase useful to the present invention includes but is not limited to come from reverse transcriptase below: HIV, HTLV-1, HTLV- 11, FeLV, FIV, SIV, AMY, MMTV, MoMuLV and other retroviruses are (see Levin, Cell 88:5-8 (1997)； Verma, Biochim Biophys Acta. 473:1-38 (1977)；Wu etc., CRC Crit. Rev Biochem. 3: 289-347 (1975))。

Connection

In preferred embodiments, nucleic acid template molecules are connected to substrate (also referred to herein as surface), by described herein right It implements single-molecule sequencing analysis.Nucleic acid template molecules are connected to surface, so that template/primer duplex can individually optics It differentiates.Substrate used in the present invention can be two dimension or three-dimensional, and may include plane surface (such as glass slide), or can be at Shape.Substrate may include glass (such as controlled pore glass (CPG)), quartz, plastics (such as polystyrene (low cross-linking and Gao Jiao Polystyrene), polycarbonate, polypropylene and poly- (methymethacrylate)), acrylic copolymer, polyamide, silicon, metal (such as alkanethiol compound (thiolate)-derivatization gold), cellulose, nylon, latex, glucan, gel-type vehicle (such as silicon Glue), polyacrolein or composite material.

Suitable three-dimensional substrates include for example: sphere, particle, pearl, film, slide glass, plate, micro Process chip, pipe (such as capillary Pipe), micropore, microfluidic device, channel, filter or any other structure suitable for locked nucleic acid.Substrate may include planar array Column, or can have the matrix in the region including template nucleic acid group or primer group.Example includes the derivative CPG of nucleosides-and polyphenyl Ethylene slide glass, derivative magnetic slide glass, the polystyrene for being grafted with poly- diethanol etc..

It is preferred that coating substrate connects so that can provide best optical treatment with nucleic acid.Also it can handle for base of the invention Bottom is to reduce background.Illustrative coating include epoxides and derivatization epoxides (such as with binding molecule, such as Oligonucleotides or Streptavidin).

Various methods can be used to be anchored or be fixed on substrate surface for nucleic acid molecules.It can be by being directly or indirectly bonded to Fixation is realized on surface.It can be bonded by covalent bond.Referring to Joos etc., Analytical Biochemistry 247:96- 101, 1997；Oroskar etc., Clin. Chem. 42:1547-1555,1996；And Khandjian, Mol. Bio. Rep. 11:107-115, 1986.It is preferred that connection is the terminal nucleotide of template or the end 5' of primer and the ring for being integrated into surface The direct amine key of oxide is closed.Also it can be bonded by non-covalent bond.For example, biotin-Streptavidin (Taylor etc., J. Phys. D. Appl. Phys. 24:1443,1991) and digoxin (Smith etc., Science with anti-digoxin It 253:1122,1992) is the common tool being anchored to nucleic acid on surface and analog.Alternatively, can be by the way that hydrophobic chain be anchored Connection is realized into lipid monolayer or bilayer.Also its for being connected to nucleic acid molecules in substrate known in the art can be used Its method.

Detection

Any detection method suitable for used type can be used.Therefore, illustrative detection method includes radioactivity inspection It surveys；Optical absorbance detection, such as UV-visible absorbance detection；Optical emitting detection, such as fluorescence or chemiluminescence.Example Such as, by simultaneously or successively scanning all or part of each substrate (depending on scan method used), substrate can be detected On extension primer.For fluorescent marker, fluorescence microscope instrument singly or line by line continuous scanning can be used Selected region in substrate, for example, Fodor (U.S. Patent number 5,445,934) and Mathies etc. (U.S. Patent number 5, 091,652) described in.It includes scanning tunneling microscope (siM) and atomic force that the device from monomolecular fluorescence, which can be predicted, Microscope (AFM).Also can be used with suitable optical device CCD camera (such as Model TE/CCD512SF, Princeton Instruments, Trenton, N.J.) scan crossing pattern (Ploem, Fluorescent and Luminescent Probes for Biological Activity (fluorescence and luminescence probe of bioactivity), Mason, T. G. is edited, 1-11 pages of Academic Press, Landon, the (1993)), such as in Yershov etc., Proc. Natl. described in Acad. Sci. 93:4913 (1996), or crossing pattern can be imaged by TV monitoring.For radiation Property signal, can be used phosphorus screen imaging device (Johnston etc., Electrophoresis, 13:566,1990；Drmanac Deng Electrophoresis, 13:566,1992; 1993).Other available commercial quotient of Image-forming instrument include General Scanning Inc., (Watertown, Mass；WWW www.genscan.com), Genix Technologies (Waterloo, Ontario, Canada；WWW www.confocal.com) and Applied Precision Inc.Institute State detection method to realize and meanwhile scan a variety of connections template nucleic acid it is particularly useful.

Many methods can be used to detect the introducing of the nucleotide of fluorescent marker in single nucleic acid molecules.Optical setup includes Near-field scan microscopy, far-field confocal microscopy, the wide visual field fall penetrate illumination, light scattering, dark field microscopy, light conversion, singly and/ Or the identification of multiphoton excitation, spectral wavelength, fluorophor identification, evanescent wave illumination and total internal reflection fluorescent (TIRF) microscopy.Always For, certain methods are related to detecting the fluorescence of laser active with the microscope equipped with camera.Suitable photon detection System includes but is not limited to photodiode and enhanced CCD camera.For example, Intensified Charge Coupled Device can be used (ICCD) camera.It is imaged the single luminescent dye molecule in the fluid near surface using ICCD camera to provide very More advantages.For example, there is ICCD optical setup, it is possible to obtain a series of images (film) of fluorogen.

Some embodiments of the invention are imaged using TIRF microscopy.TIRF microscopy is excited using total internal reflection Light is known in the art.See, for example, WWW www.nikon-instruments.jp/eng/page/products/ tirf.aspx.In certain embodiments, carry out examinations using evanescent wave illumination and TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY.It can be Hidden loss of gloss field is arranged in surface, such as the nucleic acid molecules of fluorescent marker are imaged.When laser beam is in liquid phase and solid phase (such as glass) When interface between substrate is all reflected, excitation beam only penetrates short distance into liquid.Light field will not be at reflexive interface Terminate suddenly, but its intensity with distance and index decreased.This surface electromagnetic field for being called " evanescent wave " optionally excites Fluorescent molecule near interface in liquid.Very thin hidden loss of gloss field provides low background on interface, promotes the height letter in visible wavelength It makes an uproar than lower to monomolecular detection.

Evanescent field also can be in the presence of polymerase, when template/primer complex of the nucleotide incorporation connection of fluorescent marker It is imaged when middle.Then make template/primer duplex of connection and/or the core of introducing using TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY Thuja acid is shown with single-molecule resolution.

Some embodiments of the invention use non-optical detection method, such as use nano-pore (such as protein or solid State) detection, wherein molecule individually pass through that nano-pore makes can be by record various characteristics or effect (such as capacitor or blocking Electric current) feature or variation come identify molecule (see, for example, Stoddart etc., Proc. Nat. Acad. Sci., 106: 7702, 2009；Purnell and Schmidt, ACS Nano, 3:2533,2009；Branton etc., Nature Biotechnology, 26:1146, 2008；Polonsky etc., U. S. application 2008/0187915；Mitchell and Howorka, Angew. Chem. Int. Ed. 47:5565, 2008；Borsenberger etc., J. Am. Chem. Soc., 131, 7530, 2009)；Or other suitable non-optical detection methods.

Analysis

The comparison and/or compiling of the sequence results obtained from the image stack (stack) generated as generally described above are using possible The look-up table taken into account of sequence variation (due to such as error, mutation etc.).The sequencing that will substantially obtain as described herein As a result it is compared with the lookup type table containing all possible canonical sequence plus 1 or 2 base error.

Measure maternal sample in female child nucleic acid there are situations

The method of the present invention the further quantitative or qualitative analysis to sequence data is provided with detect fetal nucleic acid there are situations, and It is unrelated with the detection ability of Y chromosome, especially for detecting the female child in maternal sample.Usually by the sequence of acquisition with Reference gene group (such as maternal gene group, male parent gene group or representative are thought to can be shown that the external standard of normal numberical range) ratio It is right.Once comparison, quantify sequence obtained just to measure the quantity of the sequence reads of corresponding each chromosome.Estimate chromosome Number provides the evidence of female child in maternal sample away from 2X normal rate, also provides the tire for representing chromosomal aneuploidy The evidence of youngster's nucleic acid.

Implementable many different types of quantitative analyses are to detect the fetal nucleic acid of female child in maternal sample There are situations.The other analysis may include copy number analysis, sparse allele calls, targeting is sequenced again, differentiation DNA is repaired Decorations (such as base of methylation or modification) and breakpoint analysis.It in certain embodiments, is the presence of detection part Y chromosome Situation does not need analytical sequence data, and the method for the present invention can be related to implement quantitative analysis as described herein to detect parent sample In product fetal nucleic acid there are situations.

It is a kind of detection in maternal sample the fetal nucleic acid of female child be related to the sequence to generation there are the method for situation The copy number of column data is analyzed.This method is related to measuring in genomic segment to be become relative to the copy number of canonical sequence information Change.Canonical sequence information can be the known maternal sample (such as cheek sample) for being free of fetal nucleic acid, or can think for representative can table The external standard of bright normal, complete karyotype numberical range.In the method, by target nucleic acid in sample (genomic DNA or its portion Point) number of computations (copy number) be compared with the number of computations referring to nucleic acid.(i.e. by the standard of normal karyotype amplifying nucleic acid It is desired) quantity, or by compared with the multiple nucleic acids of the non-target chromosome from same sample, to measure referring to quantity, institute Non- target chromosome is stated to be known or doubtful be present in sample using quantity appropriate (i.e. for autosome as diploid).Copy (U.S. is special for more elaboration such as Lapidus (U.S. Patent number 5,928,870 and 6,100,029) of number analysis and Shuber etc. Benefit number 6,214,558) shown in, the content of each of which is incorporated herein by reference with it.

Normal human genome should can the only copy number containing integer (such as 0,1,2,3 etc.), and fetal nucleic acid in sample There are the copy numbers (such as 2.1) that situation can introduce fractional value.If sequence data analysis is provided with statistically significant sexual deviation The copy number measuring assembly of (being greater than the value obtained by sampling variance, reference inaccuracy or sequencing error) expected integer value, Then maternal sample contains fetal nucleic acid.For bigger sensitivity, parent and/or male parent nucleic acid samples can be used to provide in addition Canonical sequence information.Sequence information from parent and/or male parent sample, allow to identify in maternal sample it is doubtful containing Not with maternal control sample match and/or with the copy numerical value of the fetal nucleic acid of male parent sample match, show fetal nucleic acid whereby There are situations.

Another kind detection in maternal sample female child fetal nucleic acid there are the method for situation be related to implementing it is sparse Allele calls.Sparse allele calling is that analysis is located in low cover degree DNA sequencing (being, for example, less than 1 times of coverage) The method that the single allele of polymorphic site is made a variation with comparative sample amplifying nucleic acid.Genes of individuals group usually has about 3,000,000,000 Base-pair sequence.For typical individual, about 2,000,000 sites are heterozygosis, and about 1,000,000 sites are homozygous without reference Single nucleotide polymorphism (SNP).If comparing the measurement twice in phase iso-allele site in individual, in homozygous site feelings It, will be almost consistent in 100% time under condition；Or in heterozygous sites, 50% time will almost consistent (sequencing error These numbers can slightly be reduced).If comparing the measurement twice in phase iso-allele site between Different Individual, its consistency is logical To often it reduce, this depends on the not relationship between the frequency and individual of iso-allele in group.Therefore, one group in two samples Consistent degree between the allele site of wide scope can be shown that the relationship between the individual of samples taken, and wherein relationship is closer, Consistency is higher, and (such as compared with stranger, the sample of siblings or child will be more like with the sample of individual, but ratio comes from The similitude of the second sample of the same individual is small).Fig. 1 shows two samples for coming from an individual (" itself ") and this The histogram of difference between body and two family member (" family ") samples indicates monokaryon glycosides known to different one groups of sample room The comparison of sour variant.

Can by comparing maternal sample and the only sample including mother body D NA (such as cheek sample) and/or male parent DNA, The foetal DNA in maternal sample is detected using method as described above.This method is related to low cover degree (being, for example, less than 1 times Coverage) sequence information is obtained to measure in sample with the presence or absence of fetal nucleic acid.This process employs the fact that: variant exist It is labeled in full-length genome and in publicly available database millions of.Low cover degree to divide in each comparison Analyse different groups of SNP.If people find difference in being present in a large amount of variants in maternal gene group, it is expected that fetus and Difference between the genome of his/her mother is statistically significant.In addition, compared with pure maternal sample, it is contemplated that fetus gene Similitude between group and parent DNA is statistically significant, because the half of foetal DNA is inherited from its father.

The present invention relates in the site of known (coming from existing database) or doubtful (coming from data) sequence variations, relatively come Low cover degree genomic dna sequence from both the doubtful maternal sample containing foetal DNA and pure maternal sample (is, for example, less than 1 Times coverage), and if measure the difference whether than two samples be all pure parent (i.e. without containing foetal DNA) expection Difference is high.Male parent DNA sample is not needed, but can be used for additional sensibility, male parent sample will be with pure parent sample at this time Product and doubtful both samples containing foetal DNA compare.It is statistically significant higher between doubtful sample and male parent sample Similitude can be shown that foetal DNA there are situations.

The fetal nucleic acid of another kind detection female child in maternal sample is related to implementing targeting there are the method for situation It is sequenced again.It is sequenced again see, for example, Harris (U.S. Patent Application No. 2008/0233575,2009/0075252 and 2009/ 0197257) shown in, the content of each of which is incorporated herein by reference with it.In short, selecting target before sequencing Specific section (such as passing through PCR, microarray or MIPS).It will be designed as the primer hybridized with the particular section introducing, shape At primer/template duplex.The nucleotide of primer/template duplex and polymerase and at least one detectable label is allowed, full Sufficient Template Dependent allow nucleotide to be added to primer under conditions of contact.Measure label nucleotide introducing, also measurement with Introduce the identity of the nucleotide of the nucleotide complementation in the template of nucleotide opposite sites.

After polymerization reaction, primer can be removed from duplex.Introducing can be removed via any suitable means, such as logical The temperature of elevation surface or substrate is crossed so that duplex melts, or by change buffer condition come make duplex destabilisation or its Combination.It is known in the art for melting template/primer duplex method, and in such as Molecular Cloning, a Laboratory Manual (molecular cloning: experiment guide), the 3rd addendum, J. Sambrook and D. W. Russell, It is illustrated in the 10th chapter of Cold Spring Harbor Press (2001), introduction is incorporated herein by reference.

After removing primer, template can be allowed to contact with the second primer that can hybridize with the template.In one embodiment, The identical region in the template area that second primer can hybridize with the first primer hybridization (also referred to herein as first area), shape At template/primer duplex.Then repeated polymerization is reacted, and measures the sequence of at least partly template again whereby.

The targeting of alterable height genome area is sequenced again makes the coverage in those regions is wider (such as to cover at 100 times 1 Mb when spending).Normal human genome will contain about the mononucleotide variant of 100% or about 50% frequency, however, fetal nucleic acid is deposited Other possible frequency (such as 10%, 60%, 90% etc.) will introduced.If the analysis of sequencing data provides aobvious with statistics again Write the sequence variants frequency of sexual deviation 100% or 50% (be greater than because of sampling variance, referring to obtained by inaccuracy or sequencing error and be worth) Rate set, then maternal sample contains fetal nucleic acid.

The fetal nucleic acid of another kind detection female child in maternal sample is related to implementing to consider there are the method for situation The analysis of breakpoint.Sequence break refers to the mutation type found in nucleic acid, wherein allow entire DNA section inversion, reconfigure (shuffle) it or relocates, creates the new sequence connection being not present in original series whereby.Fetal nucleus can be contained doubtful Sequence break is identified in the maternal sample of acid, and compared with parent and/or male parent control sample.Appear in maternal control sample In be not detected and/or in male parent sample undetected statistically significant quantity identification breakpoint, show fetal nucleic acid Presence.

Detect fetal abnormality

Detection maternal sample in fetal nucleic acid ability make can development evaluation fetus whether there is abnormal non-invasive diagnostic Measurement.Therefore, another aspect of the present invention fetal nucleic acid is provided in analysis maternal sample with measure fetus whether have it is abnormal non- Invasive method.The method of the present invention is related to obtaining the sample including both parent and fetal nucleic acid, implements sequencing reaction to sample To obtain the sequence information of nucleic acids in samples, compare the sequence information of acquisition and the information nucleic acid from reference gene group, whereby Whether measurement fetus has exception.In certain embodiments, reference gene group can for maternal gene group, male parent gene group or its Combination.In other embodiments, reference gene group can think indicate the numberical range of normal complete karyotype for representative External standard, such as existing HG18 ginseng shines genome at present.

It can detect a variety of genetic abnormalities according to this method, including aneuploidy (has one or more extra-chromosome Or deletion) or one or more gene known change, the gene such as CFTR, VIII factor (F8 gene), β ball Albumen, hemochromatosis, G6PD, neurofibromatosis, GAPDH, amyloid beta and pyruvate kinase.The sequence of these known genes Column and common mutations.Can detect other gene unconventionalities, for example, be related in human chromosome missing, with transposition and inversion moving or Those of sequence replicated in chromosome replication gene unconventionality, wherein the sequence characterization is to be not present in maternal inheritance substance Fetal genetic material known inherited disorder.For example, Trisomy may include part, chimeric, ring, 18,14,13,8, 6,4 etc..It can be found in OMIM disease map http://www.ncbi.nlm.nih.gov/Omim/getmorbid.cgi Know abnormal list, content is incorporated herein by reference with it.

It is heterozygosis and homozygous mutation and aneuploidy that these genetic abnormalities, which include between parent and fetal nucleic acid,.Example Such as, losing an X chromosome copy (X monosomy) causes Turner syndrome (Turner's Syndrome), and adds one 21 Number chromosome copies lead to Down syndrome.Other diseases such as edward's syndrome and Pa Ta syndrome are respectively by addition one No. 18 chromosomes and No. 13 chromosome copies cause.The method of the present invention can be used for detecting transposition, addition, amplification, transversion, inversion, Aneuploidy, polyploidy, monosomy, trisomy, No. 21 trisomys, No. 13 trisomys, No. 14 trisomys, No. 15 trisomys, 16 Number trisomy, No. 18 trisomys, No. 22 trisomys, triploidy, tetraploidy and sex chromosomal abnormality, the sex chromosomal abnormality packet It includes but is not limited to XO, XXY, XYY and XXX.

Wherein target sequence can be present in mother body D NA (heterozygosis) with a copy but be caused in fetus (homozygous) The Exemplary diseases of disease include: sickle-cell anemia, cystic fibrosis, hemophilia and tay-Sachs disease (Tay Sachs disease).Therefore, using methods described herein, people can be by mutation there are two genomes and tool with a mutation Genomic region separate.

Sickle-cell anemia is autosomal recessive disease.It is heterozygote that, which there is 9% African American in the U.S., and is had 0.2% is homozygous recessive.Recessive alleles causes the monamino acid in hemoglobin beta chain to replace.

Tay-Sachs disease is autosomal recessive disease, leads to the degeneration of nervous system.Its symptom shows after birth.It should The homozygous recessive children of allele are seldom survived more than five years old.Patient lacks manufacture N- acetyl group-hexosaminidase Ability, enzyme degradation GM2 gangliosides.

Another example is phenylketonuria (PKU), and a kind of recessive hereditary disorder, patient, which lacks to synthesize, turns phenylalanine Turn to the ability of the enzyme of tyrosine.With the product of phenylalanine in the urine and blood of the homozygous recessive individual of the allele Tired and abnormal catabolite.

Hemophilia is a kind of disease that blood cannot normally condense.Blood factor participates in blood coagulation.Think to lack normal VIII The haemophiliac of the factor has haemophilia A, and the patient for lacking the IX factor has haemophilia B.X chromosome carries these bases Cause, so sequencing approach of the invention can be used for detecting the deficiency X chromosome whether fetus inherits mother or father just Normal allele.

The gene mutation list for being applicable to this method is found in GDB human genome database http: // Www.gdb.org/gdb, for the annotation human gene established by RTI International, North Carolina USA Worldwide official's database of group.

Shown in (U.S. Patent Application No.s 2005/0164241) such as the primer of chromosome specific such as Hahn, whole passes through It is incorporated herein by reference.Gene can be prepared based on the nucleotide sequence obtained from database such as GenBank, EMBL etc. Primer.For example, there is the primer more than 1,000 No. 21 chromosome specific to be listed in the network address of NIH UniSTS, the network address are as follows:。

The importance of diagnostic assay is that the measurement distinguishes false negative (fetal nucleic acid is not detected) and true negative (in health Nucleic acid is detected in fetus) ability.The method of the present invention provides such ability by following: in test sample at least partly Y chromosome there are situations, and also implement other analysis if Y chromosome is not detected in the sample.In certain implementations In scheme, the method for the present invention distinguishes false negative and true negative, and unrelated with the detection ability of Y chromosome.

If detecting Y chromosome in maternal sample, the method for the present invention ensures that the measurement can be functioned appropriately, because It is only related to male for Y chromosome, and there are just exist in maternal sample when male fetus nucleic acid only in sample.Cause This, if exception is not detected in maternal sample, and detects at least partly Y chromosome in the sample, then pushes away with can be sure that The measurement of breaking detects fetus (because there are Y chromosomes to indicate male fetus nucleic acid in maternal sample), and fetus is not wrapped Include the genetic abnormality that the measurement is analyzed.

The method of the present invention other quantitative or qualitative analysis is also provided detect fetal nucleic acid there are situations, and with detection The ability of Y chromosome is unrelated.The step is particularly useful to the embodiment in sample including the normal nucleic acid from female child. The other quantitative analysis may include copy number analysis, sparse allele calls, targeting is sequenced again and breakpoint analysis, every It is a kind of as discussed above.Therefore, if exception is not detected in maternal sample, and there is tire to the quantitative analysis of sample announcement The presence of youngster's nucleic acid infers that the measurement detects fetus in which then can be sure that, and fetus does not include carrying out the measurement for it Genetic abnormality.

It tags

In some aspects, the method for the present invention measures whether fetus has exception by following: obtaining includes parent and fetal nucleus The maternal sample of acid；Unique label is connect with the nucleic acid in sample, wherein each label associates from different chromosome；It is right The nucleic acid of tape label implements sequencing reaction to obtain the sequence of tape label；It is to measure fetus with the sequence by quantifying tape label It is no that there is exception.

(U.S. Patent Application No.s 2008/0081330) and Steinman etc. such as label such as Kahvejian are connected on target sequence Shown in (international patent application no PCT/US09/64001), the content of each of which is incorporated herein by reference with it.Mark Label sequence generally includes the certain features for keeping the sequence useful in sequencing reaction.For example, being designed to label in the only of label Special part has at least or without homopolymer region, i.e., 2 in a column or multiple identical bases, such as AA or CCC.Also design mark It signs so as to have at least one section of editing distance when implementing base sequencing Shi Qiyu base adding order one by one, this ensures first and most The latter base not with the expection Mismatching of sequence.

Label also may include retarding agent such as chain termination nucleotide, to block the base at the end template nucleic acid molecule 3'- to add. Also label is designed to there is minimum similitude with base adding order, for example, if implementing determination of alkali base sequence one by one, usually Add a base: C, T, A and G every time in the following sequence.Label also may include at least one non-natural nucleotides, such as peptide Nucleic acid or locked nucleic acid, to enhance certain characteristics of oligonucleotides.

The unique sequences part (differentiated part) of label can be different length.The method of unique label group is designed for example It has been shown that, content are incorporated herein by reference in Brenner etc. (U.S. Patent number 6,235,475) with it.In certain realities It applies in scheme, the differentiated part of label is between about 15 nucleotide ranges of about 5 nucleotide-.In specific embodiments, label Differentiated part between about 7 nucleotide ranges of about 4 nucleotide-.Because along the uniqueness of template nucleic acid molecule measurement label Partial sequence, so oligonucleotide length should be shortest length, to read from the template nucleic acid connected with longest.It is logical Often the differentiated part of label is separated with template nucleic acid molecule and (homopolymer made to combine minimum) by least one base.

Label also includes the part as primer binding site.Primer binding site can be used to allowing existing bar shaped code mask Nucleic acid molecules hybridize with sequencing primer, and optionally the sequencing primer can be anchored in substrate.Primer binding sequence can be The unique sequences of at least two base, but its distinct order that may contain all 4 kinds of bases, usually 20-50 base is long. In specific embodiments, primer binding sequence is the homopolymer of single base, such as poly A, usual 20-70 base be long.

Label also can include retarding agent, such as chain termination nucleotide at the end 3'-.Retarding agent prevents the use when not paying attention to The end 3'- primer binding site obtains unexpected sequence information as the second sequencing primer, especially when using with dimerization When primer sequence.Retarding agent can be in any part for preventing polymerase addition base with dNTP incubation period.Illustrative resistance Stagnant dose is the nucleotide terminator for lacking 3'-OH, i.e. dideoxy nucleotide (ddNTP).Common nucleotide terminator has 2', 3'- dideoxy nucleotide, 3'- aminonucleotide, 3'- deoxynucleotide, 3' azido nucleotide, acyclonucleosides acid (acyclonucleotide) etc..Retarding agent can be connected with detectable label, such as fluorogen.It can be via unstable key example Label is connected such as disulfide bond, to determine template nucleic acid by being imaged after bar code template nucleic acid hybridizes with surface Position.Detectable label is removed usually before sequencing starts.Depending on key, pyrolysis product can be needed or can not needed Further chemical modification prevents unwanted side reaction, such as after through TCEP cracked disulfide bond, can use iodoacetamide To prevent it from generating sulfhydryl-group activity.

The method of the present invention is related to for label being connected on template nucleic acid molecule.Use various mechanical, chemistry and/or enzyme method By template nucleic acid fragmentation or desired length can be cut into, such as usually 100-500 base or longer.Can by with Lower method random shearing DNA: via ultrasonic such as Covaris method；With DNA enzymatic is of short duration contacts；Or use one or more limits The mixture of property enzyme or transposase or nickase processed.Can by with RNA enzyme is of short duration contacts, heat plus magnesium or by shearing allows RNA piece Duan Hua.Before fragmentation or after fragmentation, RNA can be converted into cDNA.

In certain embodiments, it is connect by label with template nucleic acid molecule with enzyme.Enzyme can be ligase or polymerase.Even Connecing enzyme can be any enzyme that can be connected to oligonucleotides (RNA or DNA) on template nucleic acid molecule.Suitable ligase includes T4 DNA ligase and T4 RNA ligase (ligase can be commercially available from New England Biolabs).In particular implementation In scheme, the method using ligase is known in the art.Polymerase can be that can add at the end 3' of template nucleic acid molecule Any enzyme of nucleotide.Polymerase can be for example commercially available yeast poly (A) polymerase from USB.Make according to shop instruction Use polymerase.

Connection can use complementation for flat end or via at protruding terminus (hanging end).In certain embodiments, The end of (such as using polymerase and dNTP) segment can be repaired, modifies (such as using exonuclease) or filled up after fragmentation To form flat end.When generating flat end, end disobeying with the shape end pairwise fragment 3'- can be handled with polymerase and dATP Rely the addition in template, it is prominent (overhanging) to generate single A whereby.In the method for referred to as T-A clone, this is used Single A guides the connection for having single T segment outstanding at the end 5'-.

Or, because it is known that after road restrictive digestion, possible combination outstanding is left by restriction enzyme, so leave End can be left, i.e., irregular end.In certain embodiments, using the Double stranded oligonucleotide with complementary protruding terminus Acid.In particular instances, prominent method using A:T single base (referring to Fig. 1-2).

In specific embodiments, substrate has been anchored the sequence with primer binding sequence reverse complemental in oligonucleotide, such as 5'-TC CAC TTA TCC TTG CAT CCA TCC TCT GCC CTG or poly T (50).When the sequence of same dimerization is used for When primer, implementing this field to be known as the program of " filling and leading up and lock (fill and lock) " be advantageous.When in sample When poly A (20-70) hybridizes with the poly T (50) on surface, it is likely that do not have a perfect alignment, therefore by allowing sample and poly- Synthase and TTP are incubated with to fill and lead up the hybrid.After filling and leading up step, washing sample, allow polymerase and it is one or two kinds of with The dNTP of base complementrity used in lock sequence is incubated with.Also can implement to fill and lead up and lock during single step, In this process, polymerase, TTP and one or two kinds of reversible terminators (complement of locking base) are mixed simultaneously It is incubated for.At this stage, reversible terminator terminates addition, and the processing by being specific to analog used can make the terminator (reverse of suppression mechanism) is functioned again.Some reversible terminators have functional resistance to the 3'-OH that needs are removed It is stagnant, and other reversible terminators such as Helicos BioSciences Virtual terminator have connected via disulfide bond Inhibitor in base, the disulfide bond can be removed by being handled with TCEP.

In addition the nucleic acid from maternal sample is sequenced as described herein after label.Label can make to come from different dyeing The template nucleic acid of body is distinguished from each other in entire sequencing procedure.Because each label associates from different chromosome, can Quantify sequence label.The deviation of any and 2X natural rate of interest of assessment sequence reading, the deviation show fetal abnormality.

In an alternative embodiment, by allowing the end 3' of bar code sequence and maternal DNA fragments to connect before sequencing To make bar code on cell-free maternal nucleic acids band.It is preferred that bar code is 5-8 nucleotide, it is used as parent Cell-free DNA Unique identifiers.Those sequences also may include polynucleotides (such as poly-A) tail of 50 nt.Nucleic acid then can be made by doing so Directly hybridize with flowing pool surface, is then sequenced.Among other, this method especially can be such that different maternal DNA samples joins It closes and enters for being sequenced in single flow cell channel, so that reaction can multiplex.

Detect unique sequences

In some aspects, detect fetal nucleic acid by following steps using the method for the present invention: obtaining doubtful includes fetal nucleic acid Maternal sample；At least two unique sequences in test sample；It is female with being measured based on the mutual ratio of two kinds of sequences detected It whether there is fetal nucleic acid in body sample.Unique sequences are that known primary sequence only occur in related gene group (such as people) Column can be measured for known unique k- aggressiveness (k-mer) or by sequencing.Advantageously, these methods of the invention It does not need to make comparisons with canonical sequence.In maternal sample, it is contemplated that two or more unique k- aggressiveness are gone out with identical frequency Existing, leading to its ratio is 1.0.Statistically there is significant variance to show that there are fetal nucleic acids in sample with expected ratio.

In certain embodiments, based on uniqueness k- aggressiveness obtainable in human genome recognize come measured in advance it is a kind of or A variety of unique k- oligomeric sequences.For example, being possible to estimate the quantity of uniqueness k- aggressiveness in any genome based on consensus sequence. The practical occurrence rate of the unique sequences of the readily available any given quantity base of those of ordinary skill in the related art this know Know.

In one embodiment, the number progress to any two or a variety of unique sequences are detected in maternal sample It counts.For example, sequence A (such as unique 20- aggressiveness) can be detected 80 times, sequence B (such as unique 30- aggressiveness) can be examined It measures 100 times.If in human genome or at least detected in one or more parts including sequence A and B consistent Sequence, then the fetal nucleic acid with sequence B is present in maternal sample with the level for being higher than parent background, shows the level extremely Small part ratio is (100-80) ratio 80.Just do not detect for sequence consistently, various known statistical analysis can be used Method measures whether the measurement difference between sequence A and the frequency of sequence B is statistically significant.

Also one or both of sequence A, B may be selected with content (such as rich in GC), to be based on the common skill in this field Factor known to art personnel, which makes unanimously to detect, to be more likely to.It is more credible compare statistics that a large amount of unique sequences may be selected.In addition, Sequence can be selected based on its position in genome area of special interest.For example, can be because of it in chromosome Sequence is selected in the presence of related with aneuploidy.Therefore, in certain embodiments, if based on its not in chromosome with it is non- The related position of ortholoidy and selected sequence A (detecting 80 times), and based on it is related with aneuploidy in chromosome Position and selected sequence B (detecting 100 times), then can make the diagnosis of fetus aneuploidy.

In other embodiments, unique sequences include one or more known SNP in known site.Except to parent sample It, also can be to the sequence in known SNP site with a variant (such as " G ") outside detecting that the number of sequence A is counted in product It arranges the number of A and in the SNP site there is the number of the sequence A of another variant (such as " T ") to be counted.If mother and Same base of both fetuses in the site is not homozygote, so that it may pass through one of G or T and any other zygosity of hypothesis Combined statistics may any deviation of horizontal (phase relative to any determination needs level) detect fetal signals.For mother Affine fetus is all homozygous situation in SNP site, can as previously described with the unique sequences of another or multiple measured in advance (such as sequence B) is compared.

In another method, sequence detected needs not be unique, and does not need measured in advance.In addition, being not necessarily to Know any information about people (or other) genome.On the contrary, being based on n-aggressiveness (or n- aggressiveness and k- aggressiveness etc.) mould Formula can distinguish the mark and the mark of fetus (if present) of mother.For example, may be present in the n- aggressiveness of any mode SNP, so that mother has a base (such as " G "), and fetus (if present) is at least the one of two allele There is another base (such as " T ") in a.If all n- aggressiveness are (in view of any error rate is in sufficiently large sample In) all there is " G ", then it is believed that no fetal nucleic acid.If the n- aggressiveness in some statistically significant quantity of SNP site has There is " T ", then detect fetal nucleic acid, and its quantity relative to mother's nucleic acid can be measured.Even if may be present at such two Or when many places, wherein there is (i.e. sequence is not unique) in one or both of mother or Fetal genome in n- aggressiveness, this It is correct, because in view of the reading of sufficiently large quantity, the existence or non-existence situation based on fetal signals, detected There can be statistical significant difference in SNP.That is, gene frequency (its by only one rather than two (or more It is more) work biology and detected between expected allele) there can be statistical significant difference.

It is incorporated by reference into

Present disclosure is in the whole text to other files such as patent, patent application, patent publications, periodical, books, paper, online Content made reference and quote.For all purposes thus all files are incorporated herein by reference with it.

Equivalent embodiments

In the case where not departing from spirit of that invention or its essential characteristic, the present invention can be implemented with other concrete forms.Therefore, recognize Be in all respects for foregoing embodiments illustrating property rather than limitation invention described herein.Therefore, described claims and Non- preceding description shows the scope of the invention, thus falls into all in the meaning and scope of claims equivalent embodiments Variation is intended to all be included therein.

Embodiment

Embodiment 1: measurement sample in fetal nucleic acid there are situations

The nucleic acid samples from lymphocyte are obtained from normal health adult male and women.By scheme known in the art come Extract nucleic acid.Sample setting includes 2 tri- sample sets of HapMap (trio) (6 samples), at 8 of three different machines (single-molecule sequencing instrument, Helicos BioSciences Corporation) runs (2 in HELISCOPE sequenator channel Technical repetition).To the genomic DNA sequencing from one of sample in each channel (8-13M uniqueness compares reading).

Data set includes 8 compressed files, one, each channel HELISCOPE.Sequence reads are mapped to referring to people's base Because of group, remove non-unique reading (Fig. 2) compared.Firstly, based on autosomal tale by the counting criteria of every a sample Change (Fig. 3).Then, based in all samples with the average mark of the reading of each chromosome ratio pair (chrX- only women, chrY- Only male；Fig. 4), each chromosome counting is standardized.

Data show quantitative chromosome analysis (Fig. 5).These data show that the base of selected HapMap sample Because of a group sequencing, including both male and female, and the then accurate quantitative analysis of chromosome counting.Data display identification X herein The different abilities of the expection ratio of chromosome and Y chromosome.Statistics indicate that from just derived from the genomic DNA obtained from individual The uniformity of genome covering expected from normal diploid gene group, and prove that fetal nucleic acid is not present in these samples.It is each The deviation that chromosome standardization counts is average 0.5% CV.Chromosome is bigger, and deviation is lower (0.2-0.3%), and chromosome is smaller, Deviation is higher (0.8-1.1%).Women and male sample can obviously be distinguished.

Embodiment 2: it detects the fetal nucleic acid in maternal sample and detects trisomy

The cell-free blood plasma nucleic acid of parent is obtained with method well known in the art such as Qiagen Nucleic acid purification kits.Then Nucleic acid is handled by following scheme.In short, the program by one hour 3' poly A hangover step, then one hour 3' it is bis- take off Oxygen method-blocking step composition.Implement the program with 500 pg nucleic acid.

Required reagent

Terminal enzyme (DNA) kit NEB M0315

dATP　　　　　　　　　　　　　　　Roche 11277049001

Biotin-ddATP Perkin Elmer NEL548001

The oligonucleotides of carrier oligonucleotides 50- aggressiveness

Bovine serum albumin(BSA) NEB B9001S

The water of nuclease free

Quant-iT PicoGreen dsDNA reagent Invitrogen P11495

Required instrument

Ground (milled) is used for the pre- cold aluminium block of 0.2 mL pipe

Thermal cycler

P-2, P20, P200 suction pipe

Ice bucket

For the Nanodrop 3300 of PicoGreen measurement or the plate reader of standard

Method

Before implementing DNA hangover reaction, removal RNA pollutant is digested with RNA enzyme, and react purification kit with Qiagen (catalog number (Cat.No.) 28204) purification.Accurate quantitative analysis DNA is answered before the use.Use Quant-iT PicoGreen dsDNA reagent 3300 Fluorescence Spectrometer of kit (lnvitrogen, catalog number (Cat.No.) P11495) and Nanodrop.In DNA cleaning/settling step phase Between, use the other nuclease free glycogen of molecular biology grade or linear acrylamide as carrier.

Prepare following mixture: 10 X buffer of NEB terminal enzyme (DNA) (2 μ l)；2.5 mM CoC1₂(2μl)；Parent The water (10.8 μ l) of cell-free plasma nucleic acid and nuclease free.Total volume is 14.8 μ l.Allow in the thermal cycler mixture in It is heated 5 minutes at 95 DEG C so that DNA is denaturalized.After heating, allow mixture cooling to obtain single stranded DNA on the aluminium block of pre-cooling, Aluminium block is maintained in ice and aqueous slurry (about 0 DEG C).Cool down sample as quickly as possible to prevent the single stranded DNA re-annealing of denaturation.

Following mixture is added in the DNA being denaturalized above on ice: the terminal enzyme (DNA) of 1 μ l is (with 1 times of buffering Liquid dilute 1:4 to 5U/ μ l), 50 μM of dATP of 4 μ l；With the BSA of 0.2 μ l.The volume of the mixture is 5.2 μ l, so that The total volume of reaction is 20 μ l.Pipe containing mixture is placed in thermal cycler, run following procedure: 37 DEG C 1 hour；70 DEG C 10 minutes；Temperature return is allowed to be down to 4 DEG C.Poly (A) tail is added in DNA at this time.

Make within 5 minutes the 20 μ l Polyadenylation reactant by the thermal cycler heating mixture at 95 DEG C Denaturation, then use is maintained at ice and cools down rapidly with the pre- cold aluminium block in aqueous slurry (about 0 DEG C).As quickly as possible cool down sample to prevent The single stranded DNA re-annealing being only denaturalized.

Following blocking mixture is added in the Polyadenylation mixture being denaturalized above: the terminal enzyme (DNA) of 1 μ l 10 times of buffers；The CoC1 of 1 μ l₂(2.5 mM)；The terminal enzyme (DNA) of 1 μ l (dilutes 1:4 to 5U/ μ with 1 times of buffer L), 200 μM of biotin-ddATP of 0.5 μ l；With the water of 6.5 μ l nuclease frees.The volume of the mixture is 10 μ l, so that The total volume of reaction is 30 μ l.

Pipe containing mixture is placed in thermal cycler, run following procedure: 37 DEG C 1 hour；70 DEG C 10 minutes；It allows Temperature return is down to 4 DEG C.It observes at this time to block the end 3' and be added in the DNA of poly-adenosine.

The control oligonucleotide of 2 picomoles is added in above-mentioned heat-inactivated 30 μ l terminal enzyme (DNA) reactant.It will be right It is added in sample according to oligonucleotides so that sample loads the DNA minimization of loss in step.Control oligonucleotide is without containing more Poly- (A) tail, therefore will not hybridize with flowing pool surface.Sample has been ready miscellaneous with the flow cell for sequencing reaction at this time It hands over.Without other cleanup step.

Sample is loaded into HELISCOPE sequence instrument channel (single-molecule sequencing instrument, Helicos according to shop instruction BioSciences Corporation).According to shop instruction in the channel to the DNA sequencing from sample.By sequence reads It is mapped to and removes non-unique reading compared referring to human genome.Firstly, based on autosomal tale by every a sample Counting criteria.Then, based in all samples with the average mark of the reading of each chromosome ratio pair (chrX- only women, ChrY- only male), each chromosome counting is standardized.By the chromosome counting of 1,18 and No. 21 chromosome in sample with The deviation of desired value based on control sample is compared.

Figure 10 shows the analysis result of sequence information.In the figure, No. 1 chromosome is used as control.The data of this paper Display detects foetal DNA (Figure 10).The data of this paper, which further display, also detects No. 18 chromosomes and No. 21 chromosome Trisomy (Figure 10).

The correction of embodiment 3:GC preference

It (is indicated based on correlation to quantify each chromosome or chromosome when implementing chromosome counting analysis based on sequencing information The quantity of section), it will be in the relative populations of the reading of each chromosome (or chromosome segment) and one or more normal specimens Measured standard comparing.Certain steps of sample preparation and sequencing procedure can lead to GC preference, wherein the phase of each chromosome Expression is not only influenced by the relative populations of the chromosome (copy number), but also is influenced by its G/C content.The sample of measurement The difference of GC preference can lead to the deviation of chromosome counting between product and control (normal) sample, so that there is the dye of extreme G/C content Colour solid is likely to occur more more or less than its practical copy number.Fig. 6 is that display GC preference leads to chromosome counting sample devious The diagram of product.It sorts by cumulative G/C content to chromosome.These data are shown for the chromosome with extreme G/C content, are surveyed The variability of amount is higher.

The method of the present invention can measure the amount of GC preference in sequence information obtained, and also the GC in recoverable sequence information is inclined It is good.In certain embodiments, the method for the present invention is related to obtaining nucleic acid sequence information to sample sequencing；It measures in sequence information The amount of GC preference；Correction sequence information is to explain GC preference；With the information after analysis correction.

The measurement of the amount of GC preference in sample can be realized with many modes.It in certain embodiments, can be by by base Because group is assigned to different units and to measure the correlation between the number counted in each unit and its G/C content inclined to quantify GC Good amount.Fig. 7 is the diagram for each unit for showing the function as G/C content in unit and drawing.In this embodiment, Genome is assigned in the unit of 1000 kbp.Although the number is illustrative, and any size can be used.Significantly Negative or positive correlation shows the presence of GC preference (see Fig. 7).In Fig. 7, sample above is shown to be positively correlated with G/C content, and following Sample show it is negatively correlated with G/C content.

The method of the present invention reduces or eliminates the influence of GC preference in sequence information.Many schemes can be used to reduce or eliminate The influence of GC preference in sequence information.In certain embodiments, genome units subgroup is selected in given range, so that often The average G/C content of one chromosome is balanced (or deviation is smaller).Then chromosome counting is implemented to selected subgroup.Fig. 8 is provided The embodiment of the program.In fig. 8, analysis is only limitted to the genome units that given G/C content is 0.42-0.48, that is, accounts for genome About 25% (the A group of Fig. 8).

The B group and C group of Fig. 8 is shown in the difference after the GC preference in correction sequence information in sequence information obtained. The B group of Fig. 8 shows the sequence information before the correction of GC preference.The C group of Fig. 8 shows the sequence information after the correction of GC preference.This A little data show that GC preference deviates chromosome counting, so that the chromosome with extreme G/C content seems than its practical copy number It is more or less.After the GC preference in correction sequence information, data show more accurate chromosome counting, so that detectable The trisomy of No. 18 and No. 21 chromosome, this is impossible to be obtained according to the sequence information analysis before correcting GC preference.

In other embodiments, with mathematical function (such as primary or quadratic polynomial) in one group of genome units, To the correlation modeling between G/C content and chromosome counting.Illustrative mathematical function is regression model (i.e. by sequence information It is fitted with mathematical function, such as lowfunction (linear and/or quadratic polynomial)).By being subtracted from the counting of each unit The component of the GC- dependence reflected in model corrects the influence of GC preference.Dyeing is implemented in counting after being then based on correction Body counts.The advantage of the embodiment remains the count number of raw data set for it, this sensitivity for the method It is important.

The embodiment of Fig. 9 offer program.In Fig. 9, by the GC for subtracting linear model from each genome units Dependence carrys out correction sequence information.The A group and B group of Fig. 9 shows the sequence information before the correction of GC preference.The C group and D group of Fig. 9 Show the sequence information after the correction of GC preference.These data show that GC preference deviates chromosome counting, so that having extreme The chromosome of G/C content seems more more or less than its practical copy number.After GC preference in correction sequence information, data are shown More accurate chromosome counting, so that the trisomy of detectable No. 18 and No. 21 chromosome, this is impossible according to correcting What the sequence information analysis before GC preference obtained.

In still other embodiments, GC preference corrects as follows.Obtain being averaged for each unit in many control samples Coverage, by the coverage observed in sample divided by the average value of control population (in view of control sample entirety coverage Different level, this may be weighted average).Then the cell value after each correction is the ratio of observation and desired value, should Ratio will be more consistent between the unit of different %GC.

<110>Sequenom, Inc.

<120>method for detecting fetal nucleic acid and diagnosing fetal exception

<130> HELI-133/02WO 28526/427

<140> PCT/US11/24132

<141> 2011-03-16

<150> 12/709,057

<151> 2010-02-19

<150> 12/727,824

<151> 2010-03-19

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 32

<212> DNA

<213>artificial sequence

<220>

<223>synthetic oligonucleotide

<400> 1

tccacttatc cttgcatcca tcctctgccc tg

Claims (3)

1. a kind of for obtaining the side with reduced GC preference or the sequence reads counting without GC preference of nucleic acids in samples Method, which comprises

The sequence reads obtained from the nucleic acid of sample are mapped to reference gene group unit, in which:

(i) unit represents allocated reference gene group；(ii) unit has same size；(iii) unit Represent the different sections of the chromosome of reference gene group and the G/C content of (iv) determination unit；

Determine that the sequence reads for being mapped to each unit count；

Select G/C content for the unit subgroup of 0.42-0.48, wherein the sequence reads of selected unit subgroup show that reduced GC is inclined Get well or do not have GC preference；With

The sequence reads for analyzing selected unit subgroup count.

2. the method for claim 1 wherein selected unit subgroups to represent 25% reference gene group.

3. the method for claims 1 or 2, wherein the size of unit is 1000 kilobase (kb).