Patent Application
20190048414
For example, hybridoma production technology is widely accepted as a means for preparing large amounts of monoclonal antibodies and for research and clinical applications. However, if culturing of hybridoma is continued, there is a concern that reactivity of a produced antibody is changed due to somatic mutations. For this reason, in a case of preserving a useful antibody and preparing a modified antibody, it is required to determine a nucleic acid sequence of a gene of the antibody produced by hybridoma.
In the related art, in determining a nucleic acid sequence of an antibody gene, the method of 5′ Rapid Amplification of cDNA Ends (5′ RACE), the degenerative PCR method, and the like can be used (for example, refer to NPL 1)
[NPL 1] Zhou, H., et al., Optimization of primer sequences for mouse scFv repertoire display library construction, Nucleic Acids Research, 22 (5), 888-889, 1994.
However, the 5′ RACE method requires a large amount of total RNA and is difficult to carry out in some cases. In addition, in the degenerative PCR method, there are cases where loss of the original nucleic acid sequence is caused due to mis-hybridization of degenerative primers.
An object of the present invention is to provide a method for conveniently and accurately determining a nucleic acid sequence of a target gene expressed in a subject cell.
The present invention includes the following aspects.
[1] A method for determining a nucleic acid sequence of a target gene expressed in a subject cell, including comprehensively determining mRNA nucleic acid sequences in the subject cell, and identifying a nucleic acid sequence having a nucleic acid sequence of a portion of the target gene, from among the determined mRNA nucleic acid sequences, in which the identified nucleic acid sequence is a nucleic acid sequence of the target gene.
[2] The method according to [1], in which a rank of the target gene is first to tenth in a case where the ranks of all genes expressed in the subject cell are determined in order from the largest number of mRNA molecules.
[3] The method according to [1] or [2], in which the subject cell is an antibody-producing cell, and in which the target gene is an antibody heavy chain gene and a nucleic acid sequence of a portion of the target gene is a nucleic acid sequence of a portion of a constant region of the antibody heavy chain gene, or the target gene is an antibody light chain gene and a nucleic acid sequence of a portion of the target gene is a nucleic acid sequence of a portion of a constant region of the antibody light chain gene.
[4] The method according to any one of [1] to [3], in which comprehensively determining mRNA nucleic acid sequences is performed by next generation sequencing.
[5] The method according to [4], in which the number of reads of the nucleic acid sequence in the next generation sequencing is 50,000 reads or less.
According to the present invention, it is possible to provide a new technology capable of conveniently and accurately determining a nucleic acid sequence of a target gene expressed in a subject cell.
In Embodiment 1, the present invention provides a method for determining a nucleic acid sequence of a target gene expressed in a subject cell, including a step of comprehensively determining mRNA nucleic acid sequences in the subject cell and a step of identifying a nucleic acid sequence having a nucleic acid sequence of a portion of the target gene, from among the determined mRNA nucleic acid sequences, in which the identified nucleic acid sequence is a nucleic acid sequence of a target gene.
According to the method of the present embodiment, it is possible to conveniently and accurately determine a nucleic acid sequence of a target gene. In addition, the method of the present embodiment can be carried out using only approximately 0.1 μg of total RNA. For this reason, with one subject cell as a sample, for example, a nucleic acid sequence of a target gene at one cell level can be determined.
In the method of the present embodiment, the step of comprehensively determining mRNA nucleic acid sequences is preferably performed by next generation sequencing. More specifically, the method of the present embodiment can be carried out by mRNA-seq that comprehensively determines mRNA nucleic acid sequences by the next generation sequencing.
“Next generation sequencing (NGS)” is a term that is used in comparison with a first generation sequencer represented by a fluorescent capillary sequencer using sequencing method by the Sanger method. The next generation sequencing substantially includes various machines or technologies, and it is assumed that various forms of the next generation sequencers will be designed from now on.
In the first generation sequencer, the number of specimens that can be processed at a time is limited to a maximum of approximately 96. In addition, a DNA molecule used as a sample for performing sequence determination was required to be prepared by being separately cloned in advance and amplified by the PCR method, and enormous efforts were required at that stage.
In contrast, in the next generation sequencing using the next generation sequencer, DNA fragments including various sequences are arranged in parallel and analyzed by applying amplification technologies such as emulsion PCR and bridge PCR or high-sensitivity detection technology such as one molecule observation. For this reason, larger-scale nucleic acid sequences can be conveniently determined.
Specific examples of the next generation sequencer include MiSeq, HiSeq, NovaSeq (Illumina); Genetic Analyzer V2.0, Ion Proton (Thermo Fisher Scientific); MinION, PromethION (Nanopore), and the like.
In the step of comprehensively determining mRNA nucleic acid sequences in a subject cell, library preparation is performed depending on the type of the next generation sequencer that is used. For example, sequencing may be performed at an average read length of 50 to 100 bp and a number of reads of 30,000 to 50,000. That is, the number of reads of nucleic acid sequences by the next generation sequencing may be 50,000 reads or less. As will be described later in the examples, according to the method of the present embodiment, even if the number of reads is small to that extent, a nucleic acid sequence of a target gene can be determined.
Nucleic acid sequence data obtained by sequencing obtains contigs by assembling (attaching) due to an optional technique. In the specification of the present application, “contigs” refers to longer nucleic acid sequences obtained by assembling short reads. For example, contigs are the assembled full-length mRNA nucleic acid sequences. As a result, the mRNA nucleic acid sequence in the subject cell is determined. For example, assembling can be performed by a technique such as de novo assembly which does not require a reference sequence.
Subsequently, a nucleic acid sequence having a nucleic acid sequence of a portion of a target gene is identified from among the determined mRNA nucleic acid sequences (contigs). The nucleic acid sequence identified in this way is the nucleic acid sequence of the target gene.
For example, in a case where the target gene is an antibody gene, a nucleic acid sequence of a constant region of an antibody heavy chain (Igh) and a nucleic acid sequence of a constant region of an antibody λ light chain (Igl) or an antibody κ light chain (Igk) can be used as nucleic acid sequences of a portion of the target gene. More specifically, for example, nucleic acid sequences described in Sequence Numbers 11 to 14 can be used as nucleic acid sequences of a portion of a constant region of rat Igh. In addition, for example, nucleic acid sequences described in Sequence Numbers 15 and 16 can be used as nucleic acid sequences of a portion of the constant region of rat Igl. In addition, for example, a nucleic acid sequence described in Sequence Number 17 can be used as a nucleic acid sequence of a portion of a constant region of rat Igk. In addition, for example, nucleic acid sequences described in Sequence Numbers 18 to 22 can be used as nucleic acid sequences of a portion of a constant region of mouse Igh. In addition, for example, nucleic acid sequences described in Sequence Numbers 23 to 26 can be used as nucleic acid sequences of a portion of a constant region of mouse Igl. In addition, for example, a nucleic acid sequence described in Sequence Number 27 can be used as a nucleic acid sequence of a portion of a constant region of mouse Igk.
In addition, it is possible to extract a nucleic acid sequence of the target gene more efficiently by identifying a contig having a nucleic acid sequence of a portion of the target gene and having a total length equal to or greater than the total length of the target gene.
For example, in a case where the target gene is Igh, an Igh amino acid sequence includes 400 or more amino acid residues. Here, it may be possible to identify a contig having a nucleic acid sequence having a length of 1200 bp or greater required for coding the amino acid sequence. In this way, it is possible to efficiently extract the contig of a full-length target gene.
In the method of the present embodiment, the target gene that determines a nucleic acid sequence is preferably a gene of which a rank is first to tenth in a case where the ranks of all genes expressed in the subject cell are determined in order from the largest number of mRNA molecules. It is possible to easily determine a nucleic acid sequence of the target gene having a large number of mRNA molecules and within the above range.
Or, the expression amount of the target gene is preferably 5,000 fragments per kilobase of exon per million mapped fragments (FPKM) or greater. It is possible to easily determine the nucleic acid sequence of the target gene of which an expression amount is at that degree. The upper limit of the expression amount of the target gene is not particularly limited, but in general, 30,000 FPKM or so is the upper limit in many cases.
Examples of the target gene include an antibody gene, a T-cell receptor gene, a B-cell receptor gene, and the like, but the target gene is not limited thereto.
(Antibody Gene)
For example, the subject cell may be an antibody-producing cell, the target gene may be an antibody heavy chain gene, and a nucleic acid sequence of a portion of the target gene may be a nucleic acid sequence of a portion of a constant region of the antibody heavy chain gene. Or, the subject cell may be an antibody-producing cell, the target gene may be an antibody light chain gene, and a nucleic acid sequence of a portion of the target gene may be a nucleic acid sequence of a portion of a constant region of the antibody light chain gene.
In recent years, for example, there have been cases where an antibody was produced by using an animal of which a genome nucleic acid sequence was not clear, such as skunk. In such a case, there are cases where a genome nucleic acid sequence cannot be used in a reference sequence of determination of a nucleic acid sequence. According to the method of the present embodiment, since it is possible to determine a nucleic acid sequence even in a case where a reference sequence is not present, it is possible to determine a nucleic acid sequence of an antibody gene even in such a case.
In addition, in the conventional method for determining a nucleic acid sequence of an antibody gene, it was not possible to identify only a nucleic acid sequence of a variable region. In contrast, according to the method of the present embodiment, it is possible to determine a total length of a nucleic acid sequence of the target gene also including a constant region. For this reason, as will be described later in the examples, in a case where the target gene is an antibody gene, it is possible to identify even isotypes or subclasses of an antibody. In addition, for example, it is also possible to detect a small number of mutants due to somatic mutation of an antibody gene.
(T-Cell Receptor Gene)
For example, in adoptive immunotherapy for cancer and the like, there is a demand for determining a nucleic acid sequence of a T-cell receptor. Here, for example, the subject cell may be a T cell, the target gene may be a T-cell receptor gene, and a nucleic acid sequence of a portion of the target gene may be a nucleic acid sequence of a portion of a constant region of a T-cell receptor gene.
(B-Cell Receptor Gene)
For example, the subject cell may be an immature B cell, the target gene may be a B-cell receptor heavy chain gene, and a nucleic acid sequence of a portion of the target gene may be a nucleic acid sequence of a portion of a constant region of a B-cell receptor heavy chain gene. Or, the subject cell may be an immature B cell, the target gene may be a B-cell receptor light chain gene, and a nucleic acid sequence of a portion of the target gene may be a nucleic acid sequence of a portion of a constant region of a B-cell receptor light chain gene.
(Other Target Genes)
The target gene is not limited to the above genes, and may be an optional gene. According to the method of the present embodiment, for example, regarding the optional target gene, single nucleotide variants (SNVs), single nucleotide polymorphysms (SNPs), insertion/deletion (Indel), splicing variants, and the like can be easily analyzed.
Next, the present invention will be described in detail by showing examples, but the present invention is not limited to the following examples.
[Methods and Materials]
(Cell Lines)
Hybridoma cell lines (clones HD1, HD2, HD3, and HD4) established by the inventors were used in experiments. Each hybridoma was cultured by using a hybridoma serum-free culture medium (Gibco) to which 10% fetal bovine serum (FBS) was added, 1.2% penicillin-streptomycin-glutamine (Gibco), 1 ng/mL interleukin (IL)-6 or a GIT culture medium (Waco Pure Chemical Industries) to which 1 ng/mL IL-6 was added.
(mRNA-Seq)
From each hybridoma cell line, total RNA was prepared by using a commercially available kit (form “AllPrep DNA/RNA Mini Kit”, QIAGEN). A library was prepared by using 1 μg of total RNA and a commercially available kit (form “NEBNext Ultra Directional RNA Library Prep Kit”, New England Biolabs). With the kit, it is possible to produce a library by decreasing the used total RNA to approximately 0.1 μg.
Subsequently, mRNA-seq was performed by paired-end sequencing at an average read length of 50 bp using a next generation sequencer (form “HiSeq 1500”, Illumina). For each hybridoma cell line, nucleic acid sequence data having the number of reads of 40×106 reads or more was obtained.
(mRNA-Seq Data Analysis)
The obtained reads were mapped against the inventors' custom transcriptome reference sequence. The custom transcriptome reference sequence included mouse transcripts, rat transcripts, rat immunoglobulin heavy chain (Igh) constant region, immunoglobulin λ light chain (Igl) constant region, and immunoglobulin κ light chain (Igk) constant region nucleic acid sequences.
The reads were mapped with the parameter of -t8-P-L 10000 by using BWA-MEM which is a mapping program. The program TIGAR2 was used with default settings. The expression level of each gene was quantified as fragments per kilobase of exon per million mapped fragments (FPKM).
(De Novo Transcriptome Assembly)
Total reads or subsampled reads by the program “fastq-sample” (http://homes.cs.washington.edu/˜dcjones/fastq-tools) were de novo assembled using the program “Trinity”. CPU and max-memory parameters were changed according to read numbers. For example, in a case where the number of the reads was 40×106 reads, the CPU parameter was set to 8, and the max-memory parameter was set to 52 G In addition, for example, in a case where the number of the reads was 1×106 reads, the CPU parameter was set to 2, and the max-memory parameter was set to 12G.
The Igh and Igl/Igk coding nucleic acid sequences (CDS) were extracted by filtering treatment in a case where the contigs (assembled nucleic acid sequences) contained 20-to-30-bp unique nucleic acid sequences of the Igh and Igl/Igk constant region and had proper length (in the case of Igh, more than 1200 bp, and in the case of Igl/Igk, more than 600 bp).
(RT-PCR)
Each hybridoma RNA was purified by phenol/chloroform extraction. Reverse transcription reaction was performed by using a commercially available kit (form “PrimeScript (trademark) II 1st strand cDNA Synthesis Kit”, TAKARABIO INC.). PCR was performed using an enzyme (form “KOD Plus”, TOYOBO) and a thermal cycler. PCR products were purified by gel extraction to remove non-specific products. After that, nucleic acid sequences were determined by the Sanger method. Sequence numbers of nucleic acid sequences of primers using PCR are shown in Table 1.
(mRNA-Seq Analysis of Hybridoma)
Clones HD1, HD2, HD3, and HD4 which are hybridoma cell lines established as fusion cells of rat B lymphocytes and mouse myeloma cell line SP2 were subjected to mRNA-seq. Paired-end sequencing was performed at an average read length of 50 bp.
Subsequently, each transcriptome expression level was quantified by the program BWA-TIGAR2 and ordered according to expression levels.
As a result, it was clarified that in all the hybridoma clones, the Igh and Igl/Igk coding nucleic acid sequences had an expression amount of more than 10,000 FPKM, and were ranked as the transcript having the highest expression level.
The result shows that the mRNA-seq data of hybridomas contains a sufficient number of reads to reconstruct the Igh and Igl/Igk coding nucleic acid sequences.
(Assembly of Igh and Igl/Igk Nucleic Acid Sequences of Rat Hybridoma)
Reconstruction of the Igh and Igl/Igk nucleic acid sequences was attempted by de novo transcriptome assembly of the mRNA-seq data obtained in Experimental Example 1.
First, reconstruction of a full-length transcriptome was performed from the mRNA-seq data of clone HD1 using the program Trinity. Here, filtering of the reads was not performed. The number of reads was 45,406,048 reads, and the number of obtained contigs was 58,822 contigs.
Subsequently, the Igh coding nucleic acid sequence was extracted by filtering. In the filtering, contigs having 20-to-30-bp unique nucleic acid sequences of the Igh constant region were extracted. Sequence Numbers of the nucleic acid sequences used in the filtering are shown in the following Table 2.
The full-length IgH contains 400 or more residues of amino acids. Here, a 1395-bp nucleic acid sequence was identified as the Igh nucleic acid sequence having a length of 1,200 bp or more and containing the unique 24-bp nucleic acid sequence of Ighg2b. The identified Igh nucleic acid sequence was identical to the Igh nucleic acid sequence of clone HD1 of which the nucleic acid sequence was determined by the Sanger method.
Subsequently, the Igl/Igk coding nucleic acid sequences were extracted by filtering. In the filtering, the contigs having 20-to-30-bp unique nucleic acid sequences of the Igl/Igk constant region were extracted. Sequence Numbers of the nucleic acid sequences used in the filtering are shown in the following Table 3.
The full-length IgK had 200 or more residues of amino acids. Here, a 705-bp nucleic acid sequence was identified as an Igk nucleic acid sequence having a length of 600 bp or more and containing a unique Igk nucleic acid sequence. The identified Igk nucleic acid sequence was identical to the Igk nucleic acid sequence of clone HD1 of which the nucleic acid sequence was determined by the Sanger method.
At the same time, the inventors also identified nucleic acid sequences of antibody genes produced by clone HD2 (Ighg2a/Igk), clone HD3 (Ighg2a/Igk), and clone HD4 (Ighg2a/Igk).
In addition, it was confirmed that the antibody isotypes produced by clones HD1 to HD4 are matched with the results of isotyping assay by ELISA. The above result shows that nucleic acid sequences of the Igh genes and Igl/Igk genes can be conveniently and accurately determined by de novo assembly of mRNA-seq data.
(Assembly of Igh and Igl/Igk Nucleic Acid Sequence of Mouse Hybridoma)
Similar to Experimental Example 2, Igh and Igk nucleic acid sequences of clone 8A2 and 13C7 which are mouse hybridomas were determined. Sequence numbers of the nucleic acid sequence used in filtering of mouse Igh and Igl/Igk are shown in the following Table 4.
As a result, it was confirmed that the nucleic acid sequences are matched with nucleic acid sequences of these monoclonal antibodies determined by the Sanger method, except for a region coded by degenerative primers. The result is further evidence that nucleic acid sequences of the Igh genes and the Igl/Igk genes can be conveniently and accurately determined by de novo assembly of mRNA-seq data. In addition, it was confirmed that in the method using degenerative primers, there are cases where loss of the original nucleic acid sequence occurs due to mis-hybridization of degenerative primers.
(Optimization of De Novo Assembly Conditions for Determining Nucleic Acid Sequence of Antibody Gene)
Optimization of conditions for determining nucleic acid sequences of the Igh and Igl/Igk genes was attempted using hybridoma mRNA-seq data. First, the required number of reads for determining nucleic acid sequences of antibody genes was examined.
More specifically, among total reads of mRNA-seq of each of clones HD1 to HD4, 5×103 reads, 10×103 reads, 30×103 reads, 50×103 reads, 100×103 reads, 500×103 reads, and 1000×103 reads were randomly subsampled.
Subsequently, de novo assembly was repeatedly carried out 25 times using the reads. Subsequently, the Igh and the Igl/Igk nucleic acid sequences identified using total reads were defined as correct nucleic acid sequences, and the success rate (reconstruction rate) of obtaining complete nucleic acid sequences was calculated.
The result shows that this method can accurately identify nucleic acid sequences of antibody genes from mRNA-seq data of a limited number of reads.
(Determination of Nucleic Acid Sequence of Antibody Gene Produced by Other Hybridomas)
This method for determining a nucleic acid sequence was applied to a greater numbers of hybridomas to determine nucleic acid sequences of antibody genes. As a result, in 96 or more kinds of hybridomas, it was possible to perform mRNA-seq with 200×103 reads per sample to accurately determine nucleic acid sequences of antibody genes.
According to the present invention, it is possible to provide a new technology capable of conveniently and accurately determining a nucleic acid sequence of a target gene expressed in a subject cell.
The present invention relates to a method for determining a nucleic acid sequence of a target gene. Priority is claimed on Provisional Application No. 62/302,196 provisionally filed to the United States on Mar. 2, 2016, the content of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/008320 | 3/2/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62302196 | Mar 2016 | US |
