ESTABLISHMENT AND APPLICATION OF HUMAN IMMORTALIZED B LYMPHOCYTE CELL LINE GROUP

Abstract

The present application provides a group of human immortalized B lymphocyte cell lines and use thereof, and specifically provides a combination of four closely related immortalized lymphocyte cell lines. The combination can be used as a standard product for accuracy evaluation of a detection platform. When the four closely related immortalized lymphocyte cell lines are used as standard products for epigenome, transcriptome, proteome, and metabolome, an intrinsic magnitude difference gradient can be formed to evaluate the sensitivity of histological detection.

Claims

1. A cell line for constructing a standard product for judging the accuracy of a detection platform, wherein the cell line is selected from the group consisting of: human immortalized B lymphocyte line Fudan_D5, human immortalized B Lymphocyte line Fudan_D6, human immortalized B lymphocyte line Fudan_F7, and/or human immortalized B lymphocyte line Fudan_M8; and, the deposit number of human immortalized B lymphocyte line Fudan_D5 is CCTCC NO: C2017238, the deposit number of the human immortalized B lymphocyte line Fudan_D6 is CCTCC NO: C2017253, the deposit number of the human immortalized B lymphocyte line Fudan_F7 is CCTCC NO: C2017254, and the deposit number of the human immortalized B lymphocyte line Fudan_M8 is CCTCC NO: C2017255.

2. The cell line of claim 1, wherein the cell line comprises human immortalized B lymphocyte line Fudan_D5, human immortalized B lymphocyte line Fudan_D6, human immortalized B lymphocyte line Fudan_F7 and human immortalized B lymphocyte line Fudan_M8.

3. A standard product, which is extracted from the cell line of claim 1.

4. The standard product of claim 3, wherein the standard product comprises a set of standards, and each standard is derived from the cell line of claim 1, respectively.

5. The standard product of claim 4, wherein the standard comprises a first standard, a second standard, a third standard, and a fourth standard; wherein, the first standard is extracted from a human immortalized B lymphocyte line Fudan_D5, the second standard is extracted from human immortalized B lymphocyte line Fudan_D6, the third standard is extracted from human immortalized B lymphocyte line Fudan_F7, the fourth standard is extracted from human immortalized B lymphocyte line Fudan_M8.

6. Use of the standard product of claim 3 for the preparation of a reagent and/or kit for judging the accuracy of a detection platform.

7. The use of claim 6, wherein the detection platform is selected from the group consisting of: a sequencing platform, a chip detection platform, a metabolite detection platform, a methylation detection platform, a transcriptome detection platform, a proteome detection platform, and a combination thereof.

8. A method for judging the accuracy of a detection platform to be tested, comprising the steps: (a) providing a standard product of claim 3 and a platform to be tested; (b) constructing a library for the standard product, thereby obtaining a sequencing library; (c) sequencing the sequencing library of step (b) using the platform to be tested, thereby obtaining a sequencing result; (d) comparing the sequencing result with the standard data (threshold value) of the standard sequence corresponding to the standard product, thereby obtaining the performance confirmation parameters of the method, including precision, accuracy, sensitivity, specificity, and/or detectable range, thereby evaluating and verifying the accuracy of the platform to be tested.

9. The method of claim 8, wherein in step (d), comparing the sequencing result with the standard data (threshold value) of the standard sequence corresponding to the standard product, thereby obtaining the matching value of the sequencing result and the standard data of the standard sequence, if the matching value is 99% (preferably 99.5%, more preferably, 99.974-99.980%), indicating that the sequencing platform is accurate or qualified; if the matching value is <98%, indicating that the sequencing platform is inaccurate or unqualified.

10. A kit comprising: (a) a first container, and a first standard located in the first container, the first standard being extracted from a human immortalized B lymphocyte cell lineFudan_D5; (b) a second container, and a second standard located in the second container, the second standard being extracted from a human immortalized B lymphocyte cell lineFudan_D6; (c) a third container, and a third standard located in the third container, the third standard being extracted from a human immortalized B lymphocyte cell lineFudan_F7; and (d) a fourth container, and a fourth standard located in the fourth container, the fourth standard being extracted from a human immortalized B lymphocyte cell lineFudan_M8.

Description

DESCRIPTION OF DRAWINGS

[0043] FIG. 1 shows the morphology of the immortalized B lymphocyte line (phase contrast microscope, 40).

[0044] FIG. 2 shows the results of the STR method to identify the genetic relationship between the four cell lines and the original blood sample DNA.

[0045] FIG. 3 shows the cluster diagram of the methylation level of 4 immortalized cell lines detected by the Illumina850K methylation chip.

[0046] FIG. 4 shows the cluster diagram of the mRNA expression levels of 4 immortalized cell lines detected by transcriptome sequencing.

DETAILED DESCRIPTION OF INVENTION

[0047] After extensive and intensive research and extensive screening, the inventors have unexpectedly found that 4 immortalized lymphocyte lines with genetic relationships can be combined as standard products for judging the accuracy of the detection platform, and when 4 immortalized lymphocyte lines with genetic relationship are used as standard products of epigenome and transcriptome, it can form an intrinsic magnitude difference gradient for evaluating the sensitivity of omics detection. On this basis, the inventors have completed the present invention.

[0048] Construction of Cell Lines for Standard Products Used to Identify the Accuracy of DNA Sequencing

[0049] The present invention provides a cell line for constructing a standard for identifying the accuracy of DNA sequencing. The cell line is selected from the group consisting of: a human immortalized B lymphocyte cell lineFudan_D5, a human immortalized B lymphocyte cell lineFudan_D6, human immortalized B-lymphocyte cell lineFudan_F7, human immortalized B-lymphocyte cell lineFudan_M8, and a combination thereof; and the deposit number of human immortalized B-lymphocyte cell lineFudan_D5 is CCTCC NO: C2017238, the deposit number of human immortalized B lymphocyte cell lineFudan_D6 is CCTCC NO: C2017253, the deposit number of the human immortalized B lymphocyte cell lineFudan_F7 is CCTCC NO: C2017254, and the deposit number of the human immortalized B lymphocyte cell lineFudan_M8 is CCTCC NO: C2017255.

[0050] The immortalized B lymphocyte lines Fudan_D5, Fudan_D6, Fudan_F7, Fudan_M8 of the present invention can be used for large-scale preparation of standard products of DNA, RNA, protein, and metabolites, and can also be used for studying genetic relationships.

[0051] The present invention adopts the method of transfecting human B lymphocytes with EBV to obtain a set of immortalized cell lines that can be passaged stably and retain the genetic characteristics of the original blood sample. This group of immortalized B lymphocyte lines is from the family of identical twins, Fudan_D5 and Fudan_D6 are from identical twins, Fudan_F7 is from the father, and Fudan_M8 is from the mother.

[0052] The group of cell lines of the present invention has the following characteristics:

[0053] 1) the genetic background is clear and the biological characteristics are stable. The immortalized cell line identified by the STR method is consistent with the original blood sample, the sequence characteristics of the whole genome thereof are more than 99.97% consistent with the original blood sample, and it remains stable after multiple passages.

[0054] 2) the family design of identical twins is suitable for studying the genetic relationship between offspring and parents and the corresponding omics characteristics.

[0055] Standard Product

[0056] The present invention provides a standard product, which is extracted from the cell line used for judging the accuracy of a sequencing platform of the present invention.

[0057] In another preferred embodiment, the standard product comprises a set of standards, and each standard is from the cell line according to the first aspect of the present invention.

[0058] In another preferred embodiment, the standard is selected from the group consisting of: a nucleic acid standard, a protein standard, a metabolite standard, and a combination thereof.

[0059] In another preferred embodiment, the standard comprises a first standard, a second standard, a third standard, and a fourth standard, the first standard is extracted from a human immortalized B lymphocyte cell line Fudan_D5, the second standard is extracted from a human immortalized B lymphocyte cell line Fudan_D6, the third standard is extracted from a human immortalized B lymphocyte cell line Fudan_F7, and the fourth standard is extracted from a human immortalized B lymphocyte cell line Fudan_M8.

[0060] In another preferred embodiment, the standard product is of the same type.

[0061] In another preferred embodiment, the nucleic acid standard includes a DNA standard and/or an RNA standard.

[0062] In another preferred embodiment, the DNA standard includes a first DNA standard, a second DNA standard, a third DNA standard, and a fourth DNA standard, and the first DNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_D5, the second DNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_D6, the third DNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_F7, and the fourth DNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_M8.

[0063] In another preferred embodiment, the RNA standard includes a first RNA standard, a second RNA standard, a third RNA standard, and a fourth RNA standard, and the first RNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_D5, the second RNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_D6, the third RNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_F7, and the fourth RNA standard is extracted from a human immortalized B lymphocyte cell line Fudan_M8.

[0064] Methods for Judging the Accuracy of Sequencing Platforms

[0065] The present invention provides a method for judging the accuracy of a sequencing platform, comprising the steps:

[0066] (a) providing a standard product according to the second aspect of the present invention and a platform to be tested;

[0067] (b) constructing a library for the standard product to obtain a sequencing library;

[0068] (c) sequencing the sequencing library of step (b) using the platform to be tested, thereby obtaining a sequencing result;

[0069] (d) comparing the sequencing result with the standard data (threshold value) of the standard sequence corresponding to the standard product, thereby obtaining the performance confirmation parameters of the method, including precision, accuracy, sensitivity, specificity, and/or detectable range, thereby evaluating and verifying the accuracy of the platform to be tested.

[0070] In another preferred embodiment, in step (d), compare the sequencing result with the standard data (threshold value) of the standard sequence corresponding to the standard product, thereby obtaining the matching value between the sequencing result and the standard data of the standard sequence, if the matching value is 99% (preferably, 99.5%, more preferably, 99.974-99.980%), indicating that the sequencing platform is accurate or qualified; if the matching value is less than 98%, indicating that the sequencing platform is inaccurate or unqualified.

[0071] In another preferred embodiment, the method further includes: (e) analyzing the STR of the standard product, and comparing the obtained STR analysis result with the STR analysis result corresponding to the standard product.

[0072] In another preferred embodiment, based on the comparison result of step (e), thereby obtaining the matching value between the STR analysis result and the STR analysis result of the standard product, if the matching value is 98% (preferably, 99%, more preferably, 99.5%), indicating that the sequencing platform is accurate or qualified, if the matching value is less than 96%, indicating that the sequencing platform is inaccurate or unqualified.

[0073] In another preferred embodiment, the method further includes: (f) analyzing the Mendelian error rate of the standard product, and comparing the obtained Mendelian error rate result with the Mendelian error rate result corresponding to the standard product.

[0074] In another preferred embodiment, based on the comparison result of step (f), thereby obtaining the matching value between the result of the Mendelian error rate and the Mendelian error rate of the standard product, if the matching value is 95% (preferably, 98%, more preferably, 99%), indicating that the sequencing platform is accurate or qualified, if the matching value is less than 95%, indicating that the sequencing platform is inaccurate or unqualified.

[0075] In another preferred embodiment, the method further comprises: (g) analyzing the methylation of the standard product, and comparing the obtained methylation analysis result with the methylation analysis result corresponding to the standard product.

[0076] In another preferred embodiment, based on the comparison result of step (g), thereby obtaining the matching value between the methylation analysis result and the methylation analysis result corresponding to the standard product, if the matching value is 98% (preferably, 99%, more preferably, 99.5%), indicating that the sequencing platform is accurate or qualified, and if the matching value is less than 96%, indicating that the sequencing platform is inaccurate or unqualified.

[0077] In the present invention, the platform to be tested is not particularly limited, and may be self-built by the laboratory or purchased commercially. In a preferred embodiment, the platform to be tested in the present invention is self-built by the laboratory.

[0078] The advantages of the present invention mainly include:

[0079] (1) The present invention constructs a group of family immortalized lymphocyte lines for the first time as a standard product for evaluating the accuracy of the sequencing platform. This cell line can be passaged stably and retain the genetic characteristics of the original blood sample.

[0080] (2) In the present invention, when four immortalized lymphocyte lines with genetic relationships are used as reference substances for epigenome, transcriptome, proteome, and metabolome for the first time, an intrinsic magnitude difference gradient can be formed for the evaluation of the sensitivity of omics detection.

[0081] (3) The use of the standard product in the present invention is to perform Validation on a new detection platform, laboratory, and method, and obtain a Performance Metrics, including precision, accuracy, sensitivity, specificity, detectable range, etc.

[0082] (4) The present invention provides more information for metabolite detection platform, methylation detection platform, transcriptome detection platform, and proteome detection platform.

[0083] (5) The standard product of the present invention can finally determine the threshold through data analysis on a large number of platforms and discovering the distribution law.

[0084] The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are usually based on conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are percentages by weight and parts by weight.

[0085] Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

Example 1 Establishment of Human Immortalized B Lymphocytes

[0086] The study was approved by the Ethics Committee of the School of Life Sciences, Fudan University (Lunyan Batch No. 279), and all of the 4 healthy volunteers signed an informed consent form. 15 mL of venous blood (EDTA anticoagulation) was collected from 4 volunteers for the establishment of B lymphocyte immortalization cell lines. The four healthy volunteers were born in Taizhou, Jiangsu, and belonging to a group of identical twins family.

[0087] (1) PBMC Separation

[0088] Transferring 15 mL of EDTA anticoagulated whole blood to a centrifuge tube, centrifuged at 2000 rpm for 5 minutes, and separating the plasma and blood cell layer.

[0089] The blood cells after centrifugation were diluted to 30 ml with PBS, mixed well, and slowly added to the top of the Ficoll separation solution. Centrifuged at 2000 rpm for 20 minutes. Using a pipette to first remove the upper liquid, and then carefully aspirate the white blood cells, and transfer it to a new centrifuge tube.

[0090] Adding PBS to 30 ml, mixed upside down, centrifuged, and repeated three times.

[0091] (2) Nave B Cell Sorting

[0092] Preparing Sorting buffer (PBS without calcium and magnesium ions+2% FBS+1 mM EDTA). The Nave B cell sorting test was carried out according to the operation steps of EasySep Human Nave B Cell Enrichment Kit (STEMCELL, Catalog #19254).

[0093] (3) EBV Infection

[0094] Washing the sorted Nave B cells with PBS once, counting them, and resuspending the cells with complete medium containing virus (the amount of EBV virus was 10-20% of the total volume) according to the count results, the cell number was 1105/200 L/well (48-well plate). Centrifugation and infection were performed at 2000 rpm for 1 hour, and then it was placed in an incubator for culture.

[0095] As shown in FIG. 1, the successfully transfected cells grow in suspension in clusters, in line with the typical characteristics of EBV transfected B lymphocytes.

Example 2 Identification of the Source of the Cell Line Using STR Method

[0096] Short tandem repeat (STR), also known as microsatellite DNA, is a class of DNA sequence formed by tandem repeats with 2-6 base pairs as the core unit. Since the number of core unit repeats is highly variable and abundant among individuals, it constitutes the genetic polymorphism of the STR locus. It is the second-generation genetic marker after restricted fragment length polymorphism (RFLP) and can be used for family identification of identical twins.

[0097] (1) Purification of Blood Sample DNA

[0098] Using the original blood sample of the volunteers before the establishment of the line, adding 20 L proteinase K and 50 L anticoagulant blood into a 1.5 mL EP tube, making up the total volume to 220 L with PBS, and proceeding to step 2.

[0099] Adding 200 L Buffer AL, mixed well, and placed in a 56 C. water bath for 10 min.

[0100] Adding 200 L of ethanol (96-100%) to the above sample and mixed well.

[0101] Transferring the above liquid to the purification column (DNeasy Mini spin column) and placing it in the 2 ml collection tube provided by the kit. Centrifuged at 6000 g (or 8000 rpm) for 1 min. Discarding the waste liquid and collection tube.

[0102] Putting the purification column into a new 2 ml collection tube, adding 500 L Buffer AW1, and centrifuged at 6000 g (or 8000 rpm) for 1 min. Discarding the waste liquid and collection tube.

[0103] Putting the purification column in a new 2 ml collection tube, adding 500 L Buffer AW2, and centrifuged at 20000 g (or 14000 rpm) for 3 min. Discarding the waste liquid and collection tube.

[0104] Putting the purification column in a new 1.5 ml or 2 ml centrifuge tube, adding 200 L of Buffer AE, dropping it directly on the white membrane in the middle, placed at room temperature for 1 min, and centrifuged at 6000 g (or 8000 rpm) for 1 min to elute DNA.

[0105] The eluted DNA was quantified with Nanodrop, and the sample was diluted to a concentration of 10 ng/L, and 10 L was taken for STR detection.

[0106] (2) Purification of Cell Line DNA

[0107] The Fudan_D5, Fudan_D6, Fudan_F7, Fudan_M8 cells were centrifuged at 300 g for 5 min, and resuspended in 200 L PBS solution. 20 L proteinase K was added.

[0108] Adding 200 L Buffer AL. Mixed well and placed in a 56 C. water bath for 10 min.

[0109] Adding 200 L of ethanol (96-100%) to the above sample, and make sure that the ethanol and sample were thoroughly mixed.

[0110] Transferring the liquid from the above steps to the purification column (DNeasy Mini spin column) and placed in the 2 ml collection tube provided in the kit. Centrifuged at 6000 g (or 8000 rpm) for 1 min. Discarding the waste liquid and collection tube.

[0111] Putting the purification column into a new 2 ml collection tube, adding 500 L Buffer AW1, and centrifuged at 6000 g (or 8000 rpm) for 1 min. Discarding the waste liquid and collection tube.

[0112] Putting the purification column into a new 2 ml collection tube, adding 500 L Buffer AW2, and centrifuged at 20000 g (or 14000 rpm) for 3 min. Discarding the waste liquid and collection tube. Make sure that the membrane of the purification column has been spin-dried, because residual ethanol will interfere with subsequent reactions.

[0113] Putting the purification column in a new 1.5 mL or 2 mL centrifuge tube (provided by yourself), adding 200 L of Buffer AE, dropping it directly on the middle white membrane, placed at room temperature for 1 min, then centrifuged at 6000 g (or 8000 rpm) for 1 min to elute DNA.

[0114] The eluted DNA was quantified with Nanodrop, and the sample was diluted to a concentration of 10 ng/L, and 10 L was taken for STR detection.

[0115] (3) STR Detection

[0116] Selecting 16 STR locus for identification of identical twins family, including: CSF1PO, D135317, D165539, D18551, D195433, D21S11, D2S1338, D3S1358, D5S818, D7S820, D8S1179, FGA, THO1, TPDX, vWA, and designing the corresponding primers for subsequent PCR verification.

[0117] PCR amplification: completing fluorescent PCR according to the designed experimental scheme, and using agarose electrophoresis to detect the results;

[0118] On-board detection: PCR products were detected by capillary electrophoresis using ABI 3730XL sequencing instrument to obtain data on the size of amplified fragments. Using GeneMarker v1.9 to process and analyze the collected data, converting the size and quantity of product fragments into intuitive and accurate waveform maps, and performing genetic analysis to get the results of genetic relationship.

[0119] FIG. 2 shows the results of genetic relationship of Fudan_D5, Fudan_D6, Fudan_F7, Fudan_M8 identified by the STR method. The 16 STR locus such as CSF1PO are all human genetic markers. Based on existing data and DNA analysis results, it is supported that Fudan_F7 and Fudan_M8 are the biological parents of Fudan_D5 and Fudan_D6, and Fudan_D5 and Fudan_D6 are identical twins. In addition, the DNA of the cell line is consistent with the identification results of DNA of the volunteer's original blood sample.

Example 3 Comparison of DNA from Cell Lines and Original Blood Samples

[0120] The Illumina HiSeq XTen whole-genome sequencing platform was used to perform whole-genome sequencing on the DNA of volunteers' original blood samples and cell lines, and to evaluate the influence of the establishment process on the whole genome sequence.

[0121] (1) Method of Library Constructing

[0122] The library was constructed using TruSeq Nano DNA Library Prep Kit, according to the method of TruSeq DNA Sample Preparation Guide (Illumina, 15026486 Rev. C).

[0123] (2) Sequence Determination

[0124] Using paired-end 150 bp sequencing, the average sequencing depth is 45. The sequencing reagent adopts HiSeq XTen Reagent Kit v2.5.

[0125] (3) Bioinformatics Analysis

[0126] After the quality control of the original sequencing data, bwa-mem was used for sequence alignment (mapping). The reference genome used was hg19, and the alignment results were preprocessed by GATK (mainly including eliminating the influence of excessive PCR and re-alignment and correction of indel mutations, base correction, etc.) and variant calling was performed by using HaplotypCaller. Two variant detection modes were used for each sample: conventional variant detection (VCF results, only sites that were inconsistent with the reference genome were reported) and gVCF results (all sites were reported). Then, the consistency of the original blood sample and the whole genome site of the cell line was counted. Among them, the number of inconsistent sites was counted using the VCF results of the original blood sample and cell line, and the number of detectable sites was counted using the gVCF results of the original blood sample and cell line.

[0127] The results are shown in Table 1. The consistency between Fudan_D5, Fudan_D6, Fudan_F7, Fudan_M8 and the corresponding original blood sample whole genome locus is 99.976%, 99.980%, 99.974%, 99.977%, respectively.

TABLE-US-00001 TABLE 1 Consistency results of the whole genome sequence of the immortalized cell line and the original blood sample Number of sites Number of Consistency inconsistent detectable rate Cell line with blood sample sites (%) Fudan_D5 87,505 359,719,460 99.976 Fudan_D6 87,445 427,124,551 99.980 Fudan_F7 87,660 337,853,423 99.974 Fudan_M8 87,946 386,291,050 99.977

Example 4 Effect Analysis of Quadruple Standard Product VS Triple Standard Product

[0128] In this example, in order to compare the detection effects of the quadruple standard product and the triple standard product, identical twin families were used as genomic reference substances to perform Mendelian genetic error analysis.

[0129] Methods were shown as below:

[0130] Using the Illumina HiSeq XTen whole-genome sequencing platform, whole genome sequencing was performed for the DNA of the four cell lines. The sequencing errors were evaluated according to the Mendelian genetic rules of the family, and comparing the advantages of the detection rate of sequencing errors in identical twins families over the detection rate of ordinary families of three.

[0131] (1) Method of Library Constructing

[0132] The library was constructed using TruSeq Nano DNA Library Prep Kit, according to the method of TruSeq DNA Sample Preparation Guide (Illumina, 15026486 Rev. C).

[0133] (2) Sequence Determination

[0134] Using paired-end 150 bp sequencing, the average sequencing depth is 45. The sequencing reagent adopts HiSeq XTen Reagent Kit v2.5.

[0135] (3) Bioinformatics Analysis

[0136] After the quality control of the original sequencing data, bwa-mem was used for sequence alignment (mapping). The reference genome used was hg19, and the alignment results was preprocessed by GATK (mainly including eliminating the influence of excessive PCR and re-alignment and correction of indel mutations, base correction, etc.) and the variant calling was performed by using HaplotypCaller. Two variant detection modes were used for each sample: conventional variant detection (VCF results, only sites that were inconsistent with the reference genome were reported) and gVCF results (all sites were reported). Then, the consistency of the original blood sample and the whole genome site of the cell line was counted. Among them, the number of inconsistent sites was counted using the VCF results of the original blood sample and cell line, and the number of detectable sites was counted using the gVCF results of the original blood sample and cell line.

[0137] The results are shown in Table 2. Trio1 is the Mendelian genetic error rate calculated with Fudan_D5, Fudan_F7, Fudan_M8, Trio2 is the Mendelian genetic error rate calculated with Fudan_D6, Fudan_F7, Fudan_M8, and the independently detected Mendelian genetic error rates of the Trio1 and Trio2 families of the three technical duplication families are all between 0.33 and 0.35%. In addition to the Mendelian genetic error rate of Trio1 and Trio2, the identical twin family (Quartet) can also detect inconsistent Fudan_D5 and Fudan_D6 genotypes loci, but the condition is that they meet the genetic rules of Trio1 and Trio2, respectively. A Mendelian genetic error rate of 0.9% can be detected.

TABLE-US-00002 TABLE 2 Number of detectable sites Error rate(%) Total Fudan_D5 Fudan_D6 Fudan_F7 Fudan_M8 Trio1 Trio2 Quartet Ti/Tv 1 6,503,734 4,840,979 4,836,825 4,814,322 4,827,198 0.33 0.35 0.87 1.98 2 6,512,814 4,846,926 4,846,390 4,822,709 4,843,043 0.36 0.36 0.92 1.98 3 6,512,489 4,839,350 4,847,558 4,814,966 4,835,124 0.35 0.35 0.90 1.98

[0138] The results show that it can be seen from Table 4 that when the triple standard products are used, their detection rate for Mendelian inheritance is only between 0.33 and 0.35%, and when the quadruple standard products are used, they increase the detection rate of Mendelian inheritance by about 200%, which can reflect that the data can be mutually verified by adding quadruple standard products, thereby more effectively detecting more Mendelian genetic errors. This suggests that the quadruple standard product of the present invention can provide more mutual verification information when evaluating the capabilities and detection performance of the detection platform, so as to be able to more comprehensively evaluate the performance indicators of the detection platform, including: precision, accuracy, sensitivity, specificity, detectable range.

[0139] In addition, it can be seen from the above that a synergistic detection effect can be achieved more effectively.

Example 4 Evaluation on the Quality of Methylation Detection Chip by Quadruple Standard Products

[0140] In this embodiment, when identical twin families were used as reference substances for epigenome, transcriptome, proteome, and metabolome, an intrinsic magnitude difference gradient can be formed to evaluate the sensitivity of omics detection.

[0141] In this embodiment, DNA standard products were used to detect differences in methylation of identical twin families.

[0142] Methylation Detection:

[0143] Reference substances at the epigenome, transcriptome, proteome, metabolome and other levels of identical twin families, and their intrinsic magnitude differences, that is, Fudan_D5 and Fudan_D6 were more similar in terms of epigenome, transcriptome, proteome, metabolome, etc., and the magnitude difference was smaller than the difference between that and Fudan_F7 and Fudan_M8. This inherent difference in magnitude of identical twins was innovative in evaluating the quantitative accuracy of technical platforms such as epigenome, transcriptome, proteome, and metabolome, etc.

[0144] FIG. 3 shows that the DNA samples of Fudan_D5, Fudan_D6, Fudan_F7, and Fudan_M8 are tested using Illumina Methylation 850K chip for 3 technical replicates, respectively. The Heatmap results show that from the methylation level, Fudan_D5 and Fudan_D6 have the highest similarity, and followed by Fudan_F7, Fudan_M8. This result shows that the Illumina Methylation 850K chip has high technical reproducibility and can well detect differences in methylation of identical twins.

[0145] The results in FIG. 3 show that at the DNA methylation level, three technical replicates for each of the quadruple standard product can obtain consistent clustering results, that is, Fudan_D5 and Fudan_D6 cluster first, followed by Fudan_F7 and Fudan_M8. This result shows that the quadruple standard product of the present invention has a stable intrinsic magnitude difference in DNA methylation level, and can be used to evaluate the performance of a methylation detection platform, including: precision, accuracy, sensitivity, and specificity, detectable range.

Example 5 Evaluation on RNA Transcriptome of Quadruple Standard Product

[0146] In this embodiment, the standard product of RNA was used to detect the difference in the expression level of the identical twins families.

[0147] FIG. 4 is the use of Hiseq 4000, Ribo-zero library construction method to perform 2 technical replicates detection of Fudan_D5, Fudan_D6, Fudan_F7, Fudan_M8 RNA samples, respectively. Heatmap results show that Fudan_D5, Fudan_D6 have the highest similarity, followed by Fudan_F7, Fudan_M8. This result shows that RNA-Seq has high technical reproducibility and can well detect differences in the expression levels of identical twins.

[0148] The results in FIG. 4 show that at the RNA expression level, two technical duplicates for each of the quadruple standard products can obtain consistent clustering results, that is, Fudan_D5 and Fudan_D6 cluster first, followed by Fudan_F7 and Fudan_M8. The results indicate that quadruple standard product of the present invention has stable internal magnitude differences in RNA expression levels, and can be used to evaluate the performance of the transcriptome detection platform, including: precision, accuracy, sensitivity, specificity, and detectability range.

[0149] It can be seen from Examples 4, 5, and 6, that regardless of the DNA level, DNA methylation level, and RNA level, more mutually confirmed and richer information can be provided. This suggests that it is very suitable as a standard product for a more comprehensive evaluation on testing platform, including, but not limited to: sequencing platform, chip detection platform, metabolite detection platform, methylation detection platform, transcriptome detection platform, proteome detection platform.

[0150] Culture Preservation

[0151] The human immortalized B lymphocyte cell line Fudan_D5 (the same as the deposit name) of the present invention has been deposited in China Center for Type Culture Collection on Nov. 8, 2017, address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: C2017238.

[0152] Culture Preservation

[0153] The human immortalized B lymphocyte cell line Fudan_D6 (the same as the deposit name) of the present invention has been deposited in China Center for Type Culture Collection on Nov. 8, 2017, address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: C2017253.

[0154] Culture Preservation

[0155] The human immortalized B lymphocyte cell line Fudan_F7 (the same as the deposit name) of the present invention has been deposited in China Center for Type Culture Collection on Nov. 8, 2017, address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: C2017254.

[0156] Culture Preservation

[0157] The human immortalized B lymphocyte cell line Fudan_M8 (the same as the deposit name) of the present invention has been deposited in China Center for Type Culture Collection on Nov. 8, 2017, address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: C2017255.

[0158] All literatures mentioned in the present application are incorporated by reference herein, as though individually incorporated by reference. Additionally, it should be understood that after reading the above teaching, many variations and modifications may be made by the skilled in the art, and these equivalents also fall within the scope as defined by the appended claims.