A method for measuring the translational group of a small number of cells and its application

Abstract

The present invention provides a method for measuring a small number of cell translation groups, including: 1) extracting polyribosomal mRNA of target cells of target species respectively, and sequencing to obtain mixed RNA-seq sequencing results; 2) removing homologous gene data through genome comparison analysis, and obtaining the unique read length of each target species respectively; 3) mixing the unique read lengths of two target species, and obtaining the mixed sample read length, the first species mixed read length mix read1 and the second species mixed read length mix read2 through sampling and merging; 4) respectively comparing with the genome of the corresponding species to obtain the first comparison result; 5) obtaining and identifying the mRNA being translated in the target cell through quantitative analysis and annotation of the comparison result. The present invention can identify the mRNA being translated in the target cell, and can reduce the number of sample cells required to 10 4 ~10 5 .

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics and translation group technology, and in particular to a method for identifying translation group mRNA by using mixed sequence alignment. Background Art

[0002] From gene to mRNA to protein, there are three levels of regulation, namely transcriptional regulation, translational regulation and post-translational regulation. In 2011, Schwanhausser et al. published an article in Nature magazine. Through theoretical calculations and experimental methods, they found that the impact of protein translation regulation on life far exceeds more than half of other regulation methods such as transcriptional level and mRNA degradation. It occupies the most important position in all regulation methods and is the most important regulatory step in organisms. It is well known that stem cells are closely related to the damage repair and regeneration of tissues and organs. Many studies have confirmed that protein translation plays an important role in the maintenance and differentiation of stem cell function. For example, in hematopoietic stem cells, protein translation that is too fast or too slow will significantly affect the function of hematopoietic stem cells. Tumor stem cells are the main cause of tumor recurrence and drug tolerance, which is also related to protein translation. In the skin squamous cell carcinoma model, it was found that the translation of some genes from 5'UTR led to the occurrence of tumors.

[0003] The methods for studying translational genomics using RNA sequencing include ribosome sequencing (Ribo-seq), ribosome affinity purification (RAP) or translating RAP (TRAP-seq), and polysome profiling. The mRNA bound to these ribosomes is separated to determine the protein being translated. The main advantages and disadvantages of the four technologies are as follows: Figure 2 As shown in Figure 1, the polysome profiling technique uses density gradient centrifugation to separate polyribosomes, and then sequences the RNA in the polyribosomes to identify the genes being translated ( Zhao J, Qin B, Nikolay R, Spahn CMT, Zhang G. Translatomics: The Global View of Translation. International Journal of Molecular Sciences. 2019; 20(1):212. https: / / doi.org / 10.3390 / ijms20010212). Although this method can isolate mRNA that is being translated, it requires at least a million or even tens of millions of cells. Therefore, this technology cannot be used for stem cell or primary cell research. Ribosome Nascent-chain Complex sequencing (RNC-seq) uses mass spectrometry to identify nascent peptides, but because the accuracy of mass spectrometry technology is not yet capable of resolving the sequence of nascent polypeptide chains, there is currently no method to completely detect nascent polypeptide chains. Currently, limited enzyme digestion is mainly used to study single nascent polypeptide chains. The ribosome map cannot distinguish between mRNAs bound to polyribosomes and mRNAs bound to monoribosomes, that is, it cannot identify whether they are in a state of translation. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for effectively identifying proteins in a small number of cells that are in a translation state. Using mammalian cells of different species as carriers, polyribosomes in the gradient separation solution are effectively recovered, and then the mRNA on the polyribosomes is sequenced and analyzed; using the specific sequences on the mRNA of different species, the mRNA being translated in the target cell is identified. Through this method, the conventional 10 6 -10 7 The number of cells in the sample was reduced to 10 4 , making it feasible to study the translational dynamics of stem cells or primary cells.

[0005] To achieve the above objectives, the key technology of the present invention is to utilize the specific sequences of mRNA sequences of different species to identify the translation group genes of target cells in a mixed sample.

[0006] A method for measuring the translationome of a small number of cells comprising:

[0007] 1) Extract the polyribosomal mRNA of the target cells of the two target species respectively, and sequence them to obtain mixed RNA-seq sequencing results;

[0008] 2) By comparing and analyzing the genome of the target species, the homologous gene data in the mixed RNA-seq sequencing results are removed to obtain specific reads for each target species;

[0009] 3) Mix the unique reads of the two target species, obtain mixed sample reads, the first species mixed reads mix read1 and the second species mixed reads mix read2 by sampling and merging;

[0010] 4) Aligning the first species mixed read length mix read1 with the first species genome to obtain a first alignment result;

[0011] Aligning the second species mixed read length mix read2 with the second species genome to obtain a second alignment result;

[0012] 5) The mRNA being translated in the target cell is identified by quantitative analysis and annotation of the first alignment result and the second alignment result.

[0013] In one embodiment according to the present invention, the target species are all mammals.

[0014] In one embodiment according to the present invention, the number of target cells is 10 4 ~10 5 .

[0015] In one embodiment of the present invention, in step 3), the mixing ratio of the unique reads is that the unique reads of one species are set to 10 groups with a gradient of 1% increasing from 1% to 10%, while the unique reads of another species are 100%.

[0016] In one embodiment of the present invention, in step 3), the sampling is random sampling without replacement to facilitate calculation of the proportion of the obtained mixed sample.

[0017] According to one embodiment of the present invention, in step 3), the first species mixed read length mix read1 and the second species mixed read length mix read2 are obtained by the following steps:

[0018] The read names of the unique reads of the first species and the second species are randomly sampled without replacement to obtain the sampling read name ID, and then the corresponding reads are extracted from the unique reads according to the sampling read name ID to obtain the sampling read1 and read2 of the first species and the second species, respectively. Finally, the sampled read1 of the first species is merged with the read1 of the second species, and the read2 of the first species is merged with the read2 of the second species to obtain the mixed sample reads, the mixed read1 of the first species, and the mixed read2 of the second species.

[0019] In one embodiment of the present invention, STAR is used in step 4) to align mix read1 and mix read2 with the genome, and the alignment rate and accuracy are calculated according to the grouping. When the optimal mixing ratio is finally determined to be 10:100, the alignment rate is close to the proportion of each read length, that is, almost all read lengths can be aligned, and the alignment accuracy is higher than 99%.

[0020] The present invention also provides the application of the above method in identifying mRNA in translation.

[0021] The beneficial effects of the above technical solution of the present invention are as follows:

[0022] The present invention can reduce the number of cells to 10 based on the identification of a small number of cell translation groups mRNA. 4 ~10 5 This makes it possible to study the translational group of a small number of cells, such as stem cells. At the same time, it can also be used for translational group analysis of clinical biopsy tissues to guide clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the overall framework structure diagram of the present invention;

[0024] Figure 2 References cited in this invention;

[0025] Figure 3 This is a flow chart of sequencing comparison using mixed samples in the present invention;

[0026] Figure 4 The figure is a feasibility and accuracy diagram of the method of the present invention, wherein the left one is the comparison rate statistics after mixing in different proportions, and the right one is the comparison accuracy statistics after mixing in different proportions. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0028] The present invention provides a method for simulating hybrid sequencing, such as Figure 3 , mainly including: obtaining specific reads of different species, simulating mixed sequencing results, and bioinformatics analysis.

[0029] Obtaining species-specific sequences means removing homologous genes between different species. Since homologous genes will match the reference genomes of humans and mice at the same time, thus affecting the accuracy of sequence alignment, it is necessary to remove homologous genes and obtain specific reads of each species before simulating mixed sequencing results. Obtaining specific reads is divided into three main steps.

[0030] The specific steps are as follows: First, sample processing, Polysome mRNA enrichment and purification, library construction, and sequencing are performed according to the standard sequencing process to obtain sequencing data of pure samples of different species. Then, all reads of mice and humans are matched to the reference genomes of the two species, respectively, and the read names of homologous reads of mouse and human homologous genes that match the two species are obtained according to the comparison results. Finally, "sort+uniq" is used to obtain the read names of unique reads from all reads of mice and humans, and "seqtk" is used to obtain specific reads according to the read names of unique reads. The purpose of mixing is to identify the target mRNA, so the specific reads of the two species need to be mixed in a certain ratio (the preferred ratio is mouse: human = 10:100). The simulated mixed sequencing results refer to the preparation of mixed species sequencing reads based on the specific reads of a single species. The mixing method includes two steps: sampling and merging.

[0031] The specific steps are as follows: the read names of human and mouse specific reads are randomly sampled without replacement, the sampling ratio is 1%~10% for mice (1% increase for each group), and the human ratio is 100%. The sampling read name ID is obtained according to different ratios, and then the corresponding read length is extracted from the unique read length according to the sampling read name ID to obtain the sampling read1 and read2 of human and mouse respectively. Finally, the sampled human read1 is merged with the mouse read1, and the human read2 is merged with the mouse read2 to obtain mixed sample reads, mix read1 and mix read2.

[0032] Bioinformatics analysis specifically refers to mixed sample alignment and annotation, as well as the accuracy calculation of the method:

[0033] Mixed samples mix read1 and mix read2 were aligned to the human and mouse reference genomes using STAR. The aligned results were quantitatively analyzed and annotated to obtain the measured target mRNA. Further, the alignment rate and accuracy of the mixed sample alignment results were calculated. It was found that when the ratio of mouse to human was 10:100, the alignment rate of the two species was close to the mixed ratio of the reads content of each species, and the alignment accuracy could reach more than 95%. This shows that the mixed sample alignment results are reliable, such as Figure 4 shown.

[0034] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for measuring the translational group of a small number of cells, characterized in that include: 1) Extract the polyribosomal mRNA of the target cells of the two target species respectively, and sequence them to obtain mixed RNA-seq sequencing results; 2) By comparing and analyzing the genome of the target species, the homologous gene data in the mixed RNA-seq sequencing results are removed to obtain specific reads for each target species; 3) Mixing the unique reads of the two target species, and obtaining mixed sample reads by sampling and merging, wherein the mixed sample reads include the mixed read 1 of the first species and the mixed read 2 of the second species; The first species mixed read length mix read1 and the second species mixed read length mix read2 are obtained by the following steps: The read names of the unique reads of the first species and the second species are randomly sampled without replacement to obtain the sampled read name IDs, and then the corresponding reads are extracted from the unique reads according to the sampled read name IDs to obtain the sampled read1 and read2 of the first species and the second species, respectively. Finally, the sampled read1 of the first species is merged with the read1 of the second species, and the read2 of the first species is merged with the read2 of the second species to obtain the mixed sample reads, the mixed read1 of the first species, and the mixed read2 of the second species; 4) Aligning the first species mixed read length mix read1 with the first species genome to obtain a first alignment result; Aligning the second species mixed read length mix read2 with the second species genome to obtain a second alignment result; 5) The mRNA being translated in the target cell is identified by quantitative analysis and annotation of the first alignment result and the second alignment result.

2. The method according to claim 1, characterized in that The target species are all mammals.

3. The method according to claim 1, characterized in that The target cell number is 10 4 ~10 5 .

4. The method according to claim 1, characterized in that In step 3), the unique read lengths are mixed to set the unique read lengths of the first species to 10 groups with a gradient of 1% increasing from 1% to 10%, and the unique read length of the second species is 100%.

5. The method according to claim 1, characterized in that In step 3), the sampling is random sampling without replacement to determine the proportion of the obtained mixed sample.

6. The method according to claim 1, characterized in that In step 4), STAR is used to align mix read1 and mix read2 with the genome respectively.

7. Use of the method according to any one of claims 1 to 6 in identifying translating mRNA.

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