Method and kit for determining the full length of RHD and RHCE mRNA based on third-generation sequencing

Abstract

This invention discloses a method and kit for determining the full-length RHD and RHCE mRNA based on third-generation sequencing, belonging to the field of biotechnology. It addresses the limitations of existing technologies in determining the full-length RHD and RHCE mRNA, detecting low-level splice variants, and relatively quantifying transcripts. The method includes: mRNA extraction and cDNA reverse transcription; PCR amplification; third-generation sequencing library construction and sequencing; sequencing data filtering and quality control; and transcript expression analysis. The kit includes primers for simultaneously amplifying the full-length RHD and RHCE transcripts. This invention successfully utilizes long-chain single-molecule sequencing technology to sequence the full-length transcripts of the RHD and RHCE genes, detecting multiple transcripts of the RHD and RHCE genes in RhD-positive and Del-type samples, and enabling relative quantification of the expression levels of different transcripts, providing a new method for revealing the molecular mechanisms of Rh blood type variations. The simultaneous detection method of this invention can obtain results for two genes in one step, simplifying the experimental procedure and enriching the detection results.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method and a kit for determining the full length of RHD and RHCE mRNA based on third-generation sequencing. Background Art

[0002] The Rh blood group system is one of the most polymorphic blood group systems among the known human red blood cell blood group systems published by the International Society of Blood Transfusion (ISBT). 56 antigens have been discovered, and its importance is second only to the ABO blood group system. The Rh blood group system includes two proteins (RhD and RhCE), both of which are composed of 417 amino acids and contain 12 transmembrane domains. Its encoding genes RHD and RHCE are located on chromosome 1 and are tightly linked. The RHD and RHCE genes each have 10 exons, with highly homologous sequences and up to 97% identical bases. RHD and RHCE are codominant and expressed on the surface of red blood cells at the same time. Both the RHD and RHCE genes have many alleles, resulting in multiple variants of the related antigens. There are many mechanisms for producing polymorphism, such as gene deletion or insertion, unequal gene exchange, splice site mutation, gene recombination, and gene hybridization. Some RH variants have no mutations in the coding region, but the protein expression level or epitope has changed. The mechanism may be related to the polymorphism of the non-coding region and expression regulation. Studies have pointed out that abnormal mRNA splicing and intron retention may affect D antigen expression.

[0003] In the prior art, the technical means for detecting the expression and splicing of RHD include qPCR, first-generation sequencing and second-generation sequencing. The qPCR method is only suitable for quantification and cannot effectively analyze various splicing bodies. The use of Sanger sequencing to study splicing bodies is extremely labor-intensive and difficult to detect low-abundance transcripts. The second-generation sequencing methods that have been reported so far only analyze the splicing characteristics of some regions of RHD mRNA, and there are no reports on methods for analyzing the full length of RHD mRNA. This may be because the second-generation sequencing technology needs to fragment the transcripts and then sequence the short fragments during the library construction process, resulting in difficulties in accurately assembling different transcripts, identifying variable splicing, and quantifying isoform expression. In addition, although the prior art discloses a method for DNA sequencing of the RHCE gene, there are no reports on the analysis of the RHCE mRNA sequence.

[0004] Therefore, providing a method for determining the full length of RHD and RHCE mRNA, which can not only determine the full length of RHD and RHCE mRNA, but also perform relative quantification of various splicing forms, has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining the full length of RHD and RHCE mRNA based on third-generation sequencing, so as to solve the problems in the prior art that the full length of RHD and RHCE mRNA cannot be determined, low-level splicing bodies cannot be detected, and transcripts cannot be relatively quantified.

[0006] Another object of the present invention is to provide a kit for full-length sequencing of RHD and RHCE mRNA.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a method for determining the full length of RHD and RHCE mRNA based on third-generation sequencing, comprising the following steps:

[0009] S1. Extraction of mRNA and reverse transcription of cDNA: mRNA of different samples is provided and then reverse transcribed into cDNA;

[0010] S2. PCR amplification: design primers that can simultaneously amplify the full-length mRNA of RHD and RHCE, and use the primers to amplify each cDNA obtained in step S1;

[0011] S3. Construction of third-generation sequencing library and sequencing: The PCR product obtained in step S2 is purified, quantified, end-repaired, ligated with tags, and purified again after adding sequencing adapters to complete the construction of the library; the cDNA sequencing library is sequenced on a third-generation sequencing platform;

[0012] S4. Sequencing data filtering and quality control The short fragments and low-quality data obtained by sequencing were sheared and filtered, and the 50 bp adapter sequence was removed to obtain valid data. The filtering conditions were 200 bp < measured sequence length < 3 000 bp, and sequencing quality Q score > 7. Finally, the filtered valid data were quality controlled to obtain the full-length information of RHD and RHCE mRNA.

[0013] Some embodiments of the present invention further include a step of analyzing the expression level of the transcript, specifically:

[0014] S5. Compare the measured transcripts with the human reference genome GRCh38 (hg38) gtf file in the Ensembl database; use the number of transcripts per kilobase and per million mapped reads TPM as indicators to measure the transcript or gene expression level. The TPM calculation formula is as follows:

[0015] TPMi=(Ni / Li)*1000000 / sum(Ni / Li+……+Nm / Lm)

[0016] TPMi: TPM of gene i;

[0017] Ni: number of reads on gene i;

[0018] Li: the sum of exon lengths of gene i;

[0019] sum(Ni / Li+…+Nm / Lm) is the sum of the values ​​of all (m) exons after being standardized by length;

[0020] Quantify the TPM and NumReads of each transcript for each sample.

[0021] The complete open reading frames of RHD and RHCE gene mRNA are both 1254 nucleotides, with a similarity of up to 97%. Previous studies have shown that due to the high homology of exon8 and exon10, it is impossible to exclude the interference of RHCE to design specific amplification primers only for the full-length mRNA of RHD, so the specificity can only be guaranteed by the segmented design method. However, the segmented design primer amplification of mRNA may miss the variable splicing site and miss some transcripts. The present invention adopts a third-generation sequencing method, based on the principle of single-molecule long-chain sequencing of nanopore (Nanopore), and can sequence the entire full-length transcript, and can accurately identify multiple isomers of each gene in one detection, which not only improves the sensitivity of detection, but also can accurately quantify the relative expression of each transcript of RHD and RHCE genes at the same time.

[0022] In some embodiments of the present invention, in S2, among the RHD / RHCE mRNA full-length amplification primers, the forward primer is located at the junction of the 5' non-coding region and the first exon of the RHD / RHCE gene, and the reverse primer is located at the 3' non-coding region of RHD / RHCE, which can simultaneously amplify the full-length transcripts of the mRNA of both the RHD and RHCE genes.

[0023] In some embodiments of the present invention, the nucleotide sequence of the primer is:

[0024] Upstream: 5′-GCACAGAGACGGACACAGGATG-3′;

[0025] Downstream: 5′-GCAGGCAACAGTGAGAGGAAGT-3′.

[0026] In the prior art, RHD mRNA segmented amplification is generally performed in segments 1-3, 3-9. If the third exon of the RHD gene is deleted, mutated, or the Exon3 of the transcript is deleted, these transcripts may be missed. The forward primer of the present invention is located at the junction of the 5' non-coding region and the first exon of the RHD / RHCE gene, and the reverse primer is located in the 3' non-coding region, which can specifically amplify the RHD and RHCE genes at the same time, and such deletions or missed detections will not occur.

[0027] In some embodiments of the present invention, in S2, the composition of the 25 μL amplification system is:

[0028] cDNA 2μL, 5× PrimeSTAR GXL Buffer 5μL, PrimeSTAR GXL DNA Polymerase 0.5μL, forward and reverse primers 1μL each, and ultrapure water 13.5μL to make up the volume;

[0029] The amplification conditions were 95.0°C for 3 min, 98.0°C for 10 s, 63.0°C for 15 s, 68.0°C for 1 min for a total of 35 cycles, and 72.0°C for 5 min.

[0030] In some embodiments of the present invention, in S3, the PCR product is purified by enzyme and then quantified by Qubit fluorometer; the library is constructed using Nanopore's SQK-LSK109 kit, firstly DNA end repair, A tail and Barcode label are added to connect each sample, and the library is pooled after purification by AMPure XP magnetic beads, and sequencing adapters are added, and finally AMPure XP magnetic beads are purified again to complete the library construction.

[0031] In one embodiment of the present invention, in S3, 75 μL of the cDNA sequencing library is vertically added dropwise into the sample well of the NanoporeMinION sequencing chip SpotON, and then the entire full-length transcriptome sequencing is controlled and monitored by MinKNOW software.

[0032] The present invention provides a kit for full-length sequencing of RHD and RHCE mRNA, which includes RHD / RHCE cDNA amplification primers, wherein the forward primer is located at the junction of the 5' non-coding region and the first exon of the RHD / RHCE gene, and the reverse primer is located at the 3' non-coding region of RHD / RHCE, and the full-length transcripts of the mRNA of the two genes RHD and RHCE can be amplified simultaneously.

[0033] In some embodiments of the present invention, the nucleotide sequence of the primer in the kit is:

[0034] Upstream: 5′-GCACAGAGACGGACACAGGATG-3′;

[0035] Downstream: 5′-GCAGGCAACAGTGAGAGGAAGT-3′.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention successfully utilizes the third-generation sequencing technology and performs full-length sequencing of the RHD and RHCE gene transcripts based on the characteristics of its long-chain single-molecule sequencing. The full-length sequences of all transcripts of the two genes can be obtained in one experiment, simplifying the detection steps; multiple isomers of each gene can be accurately identified, which not only improves the detection sensitivity, but also can simultaneously obtain information on the expression levels of various transcripts of the RHD and RHCE genes, and can perform relative quantification of the expression levels of different transcripts, providing a new method for further revealing the molecular mechanism of RH blood type variation.

[0038] The present invention is applied to the detection of transcripts of RhD-positive and Del-type RHD and RHCE genes, and it is found that the shearing patterns of the two phenotype RHD transcripts are extremely different.

[0039] The present invention uses the ultra-long read technology of the Nanopore platform to include a single complete transcript sequence information. This experimental sequencing obtained a total of 40,804 valid reads, with a total base number of 28,156,345bp. Due to its sufficiently high throughput, accurate expression analysis can be performed at both the gene level and the transcript level. No assembly is required for subsequent analysis, and what is measured is what is obtained, making the results more scientific and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Attached Figure 1 The gel imaging electrophoresis diagram of part of the cDNA after PCR amplification in Example 1; wherein DL2000DNAMarker (MD101-02-AA, Vazyme, China); the left diagram is RhD positive specimens 2 and 3, and the right diagram is Del specimen 1; the ag bands in the diagram represent the transcripts of the RHD and RHCE genes.

[0041] Attached Figure 2 Figure 1 is the Sanger sequencing sequence diagram of each band after cDNA amplification of RhD positive sample No. 2, gel excision, purification, and sequencing; af corresponds to the af bands in the gel imaging electrophoresis diagram.

[0042] Attached Figure 3 The expression difference of each transcript of RHD gene in Del type and RhD wild type, where *P<0.05; **P<0.01; ***P<0.001.

[0043] Attached Figure 4 The expression difference of each transcript of RHCE gene in Del type and RhD wild type, where *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1

[0047] This embodiment discloses a method for determining the full length of RHD and RHCE mRNA based on third-generation sequencing of the present invention, specifically:

[0048] 1 Materials and methods

[0049] 1.1 Research subjects Randomly selected 5 samples with RhD positive serological results and 5 samples with Del type. The RHD genotyping results of Del samples were 4 heterozygous for RHD (1227G>A) and exon 1-10 deletion, and 1 homozygous for RHD (1227G>A) / RHD (1227G>A). The RHCE genotyping results of 5 RhD positive samples were CCee in 3 cases and CcEe in 2 cases, and the typing results of 5 Del type samples were Ccee in 4 cases and CCee in 1 case. 10mL of fresh EDTA anticoagulated peripheral venous blood was collected for all samples.

[0050] 1.2 Reagents and Instruments Reagent (135306, Ambion, USA); Genomic DNA Extraction Kit (116777, Promega, USA); iScript cDNA Synthesis Kit (1708891, Bio-rad, USA); GXL DNA polymerase (R050A, TAKARA, China); AgencourtAMPureXP magnetic beads (A63880, Beckman, USA); BigDye Terminator v3.1 cycle sequencing kit (00813913, AB1, USA); TIANgel gel recovery kit (W0202, TIANGEN, China); ligation sequencing kit (SQK-LSK109, Oxford Nanopore Technologies, UK), R9 sequencing chip (FLO-MIN106, Oxford Nanopore Technologies, UK), third-generation sequencer (MinION, OxfordNanopore Technologies, UK); PCR amplification instrument (GenAmp9700, ABI, USA); first-generation sequencer (3730xl, Thermo Fisher, USA); gel imager (ChemiDoc, Bio-rad, USA); fully automatic nucleic acid extraction instrument ( 16, Promega Corporation, USA); Fluorometer (Qubit TM 4Fluorometer, ThermoFisher, USA).

[0051] 1.3 DNA extraction and RHD and RHCE genotyping Genomic DNA was prepared from whole blood samples using a genomic DNA extraction kit. The operation process was strictly in accordance with the reagent instructions, and the NanoDrop2000 UV spectrophotometer was used to detect DNA purity and concentration. Sequence-specific primer PCR reaction (sequence specific primer, PCR-SSP) was used to genotype the RHD and RHCE genes. The presence of a deletion in the RHD allele was identified by detecting the upstream rhesuse box (UP) and the hybrid rhesus box (HY). After amplification, imaging was performed after 1.5% gel electrophoresis at 110V for 20 minutes and the results were recorded.

[0052] 1.4 Extraction of mRNA and reverse transcription of cDNA The buffy coat and nucleated red blood cells were separated by Ficoll density gradient centrifugation within 48 hours of blood sample collection. After washing, 1 ml of TRIzol was added and mixed thoroughly. RNA was extracted using chloroform and isopropanol and reverse transcribed into cDNA using the iScript cDNA Synthesis Kit. The purity and concentration of RNA and cDNA were detected by NanoDrop2000 UV spectrophotometer.

[0053] 1.5 PCR amplification and Sanger sequencing Primer Premier6 software was used to design the RHD / RHCE gene mRNA primers (commissioned to Shanghai Sangon Biotechnology Co., Ltd. for synthesis). The forward primer was located at the junction of the 5' non-coding region and the first exon of the RHD / RHCE gene, and the reverse primer was located at the 3' non-coding region of the RHD / RHCE gene.

[0054] Primer sequence upstream: 5′-GCACAGAGACGGACACAGGATG-3′;

[0055] Downstream: 5′-GCAGGCAACAGTGAGAGGAAGT-3′.

[0056] The amplification system was 25 μL (containing 2 μL cDNA, 5 μL 5× PrimeSTAR GXL Buffer, 0.5 μL PrimeSTAR GXL DNA Polymerase, 1 μL each of forward and reverse primers, and 13.5 μL ultrapure water to make up the volume); the amplification conditions were 95.0°C for 3 min, 98.0°C for 10 s, 63.0°C for 15 s, 68.0°C for 1 min for a total of 35 cycles, and 72.0°C for 5 min.

[0057] After amplification, the image was taken after 1.5% gel electrophoresis at 110V for 30 minutes. The target band was cut under UV light, purified with an agarose gel DNA recovery kit, and then directly sequenced with BigDye for Sanger sequencing. The sequencing results were compared with the standard sequences on Genebank using SnapGene software, and a sequence with normal peaks in the sequencing graph was randomly selected and entered into NCBI's BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to verify whether the amplified gene was the target gene.

[0058] 1.6 Construction of third-generation sequencing library and sequencing machine PCR products were purified with exonuclease I and quantified with Qubit fluorescence instrument. The library was constructed with reference to Nanopore's SQK-LSK109 kit. First, DNA end repair was performed to add A tail and Barcode label to connect each sample. After purification with AMPure XP magnetic beads, the library was pooled and sequencing adapters were added. Finally, AMPure XP magnetic beads were purified again to complete the library construction. 75 μL of the above cDNA sequencing library was vertically added dropwise to the sample well of the Nanopore MinION platform sequencing chip SpotON, and then the entire full-length transcriptome sequencing was controlled and monitored by MinKNOW software.

[0059] 1.7 Sequencing data filtering and quality control The raw data format of the Nanopore MinION sequencer is the FAST5 format for storing and organizing large amounts of data. The Guppy software in the MinKNOW software package is used to convert the raw data from current signals to base sequence information, perform basecalling and barcode splitting, and convert the FAST5 format data to FASTQ format. First, the output data is quality controlled using EPI2ME and NanoPlot software, and then the NanoFilt software is used to cut and filter the short fragments and low-quality data in the above FASTQ format file, and remove the 50bp adapter sequence to obtain valid data (Clean data). The filtering conditions are that the measured sequence length is >200bp, <3000bp, and the sequencing quality Q score is >7. Finally, the EPI2ME and NanoPlot software are used again to quality control the filtered valid data.

[0060] 2 Results

[0061] 2.1 PCR amplification and Sanger sequencing results The OD260 / 280 of all sample RNAs was between 1.8 and 2.1, and the purity of RNA and cDNA was good. The electrophoresis results of samples with different Rh phenotypes after amplification showed multiple clear bands of different lengths (see attached Figure 1 The sequences obtained by gel excision and purification of each band and Sanger sequencing are all RHD and RHCE genes (as shown in the attached Figure 2 As shown), and the BLAST query results are all RHD or RHCE transcripts, indicating that the primers of the present invention can only specifically amplify RHD and RHCE genes.

[0062] 2.2 Quality control and analysis of full-length transcriptome data from third-generation sequencing Nanopore MinION platform sequencing showed that a total of 40,804 valid reads were analyzed, generating a total of 28.2Mb of valid data, with an average quality Qscore of 12.3, and average sequence length and N50 read length of 632bp and 722bp, respectively. After NanoFilt shearing and filtering, the number of reads was 27,132, with 20Mb of valid data, average sequence length and N50 read length of 761bp and 822bp, respectively, and the average quality Q score increased to 12.6.

[0063] Example 2

[0064] This example discloses an analysis of the expression level of the transcript obtained in Example 1, specifically:

[0065] According to Example 1, the measured transcripts are compared with the Ensembl database human reference genome GRCh38 (hg38) gtf file. Transcriptome sequencing is equivalent to a random sampling process. In order to allow the number of sequenced reads to more truly reflect the transcript expression level, the number of reads read needs to be normalized. The present invention uses the number of transcripts per thousand bases and per million mapped reads (Transcripts per million, Transcripts Per Kilobase ofexon model per Million mapped reads, TPM) as an indicator to measure the transcript or gene expression level, and the TPM calculation formula is as follows:

[0066] TPMi=(Ni / Li)*1000000 / sum(Ni / Li+……+Nm / Lm)

[0067] Ni: number of reads on gene i;

[0068] Li: the sum of exon lengths of gene i;

[0069] sum(Ni / Li+…+Nm / Lm) is the sum of the values ​​of all (m) exons after being standardized by length).

[0070] Salmon software was used to quantify the TPM and NumReads of each transcript in each sample. To analyze the difference in transcript expression between the two phenotypes, the means between multiple samples were compared using T test, and P < 0.05 was considered statistically significant.

[0071] The present invention detected 10 different RHD gene mRNA transcripts including full-length transcripts and 9 RHCE mRNA transcripts in RhD-positive, Del-type samples. The specific information is shown in Table 1.

[0072] The same transcript types were found in the RHCE gene of RhD-positive and Del-type samples. RHD-201 (full length), RHD-202 (Del89+170), RHD-203 (Del89), RHD204 (Del79), RHD-205 (Del9+170), RHD-206 (Del789), RHD-208 (Del9), and RHD-210 (Del7) transcripts were common to RhD-positive and Del-type samples. After the quantitative results were corrected, the relative expression of each transcript was shown in Figure 3 and Figure 4As shown, there were significant differences in the mean TPM values ​​of RHD-201 (full length), RHD-206 (Del789), and RHD-207 (Del8910+213) transcripts between the two phenotypes (P<0.05), but no significant differences in the mean TPM values ​​of the RHCE gene.

[0073] Table 1 Different RHD mRNA transcripts detected by third-generation sequencing of RhD-positive and Del-type samples ( n=5)

[0074]

[0075]

[0076] * aa =Amino Acid

[0077] Table 1 RhD positive, D e Different RHCE detected by three-generation sequencing of type l samples m RNA transcripts ( n =5)

[0078]

[0079] * aa =Amino Acid

[0080] In summary, the method of the present invention can sequence the entire full-length transcript and accurately identify multiple isoforms of each gene, which not only improves the sensitivity of detection, but also can accurately quantify RHD,

[0081] Relative expression level of each transcript of RHCE gene.

[0082] This experimental sequencing yielded a total of 40,804 valid reads, with a total base number of 28,156,345 bp. Due to its sufficiently high throughput, accurate expression analysis can be performed at both the gene level and the transcript level.

[0083] No assembly is required for post-analysis, what is measured is what is obtained, and the results are more scientific and reliable.

[0084] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A method based on third-generation sequencing RHD、RHCE The method for determining the full length of mRNA is characterized by: The assay method is used to determine RHD , RHCE The full length of mRNA and the expression level of transcripts are analyzed, and the determination method comprises the following steps: S1. Extraction of mRNA and reverse transcription of cDNA: mRNA of different samples is provided and then reverse transcribed into cDNA; S2. PCR amplification: Designed to amplify RHD、RHCE The full-length mRNA amplification primer is used to amplify the cDNA obtained in step S1; the nucleotide sequence of the primer is: Upstream: 5′-GCACAGAGACGGACACAGGATG-3′; Downstream: 5′-GCAGGCAACAGTGAGAGGAAGT-3′; S3. Construction of third-generation sequencing library and sequencing: The PCR product obtained in step S2 is purified, quantified, end-repaired, ligated with tags, and purified again after adding sequencing adapters to complete the construction of the library; the cDNA sequencing library is sequenced on a third-generation sequencing platform; S4. Sequencing data filtering and quality control: Short fragments and low-quality data obtained by sequencing were sheared and filtered, and the 50bp adapter sequence was removed to obtain valid data. The filtering conditions were 200 bp < measured sequence length < 3000bp, and sequencing quality Qscore > 7. Finally, the filtered valid data was quality controlled to obtain RHD , RHCE mRNA full-length information; The determination method also includes analysis of transcript expression, and the specific steps are: S5. Compare the measured transcripts with the human reference genome GRCh38 (hg38) gtf file in the Ensembl database; use the number of transcripts per kilobase and per million mapped reads TPM as indicators to measure the transcript or gene expression level. The TPM calculation formula is as follows: TPMi=(Ni / Li)*1000000 / sum(Ni / Li+……+Nm / Lm) TPMi: TPM of gene i; Ni: number of reads on gene i; Li: the sum of exon lengths of gene i; sum(Ni / Li+…+Nm / Lm) is the sum of the values ​​of all m exons after being standardized by length; Quantify the TPM and NumReads of each transcript for each sample.

2. A method based on third-generation sequencing according to claim 1 RHD、RHCE The method for determining the full length of mRNA is characterized by: In S3, the PCR product is purified with an enzyme and then quantified with a Qubit fluorometer; the library is constructed using Nanopore's SQK-LSK109 kit, and each sample is first connected by DNA end repair, A tail, and Barcode labeling, and then the library is pooled after purification with AMPure XP magnetic beads, and sequencing adapters are added, and finally the library is purified again with AMPure XP magnetic beads to complete the library construction.

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