CN114171121A - Rapid detection method for mRNA5 '3' terminal difference - Google Patents
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Abstract
The invention discloses a rapid detection method for mRNA5 '3' terminal difference. The method comprises the steps of firstly utilizing HISAT2 to compare read measured by a control group and an experimental group to a GRCh38 reference sequence, selecting fragments of intergenic regions for subsequent analysis through sequence annotation information, then utilizing overlapping sequences among the read to assemble the read one by one to form a5 'or 3' extension sequence of a gene, then forming an isotype transcript according to mRNA5 'or 3' extension sequence of the gene, comparing the experimental group and the control group, and if the requirement that the length of the experimental group is longer than the length of the control group by more than 200nt and the length of the experimental group is longer than the length of the control group and the requirement that the length of the experimental group is longer than the length of the control group and the length of the experimental group is equal to KM FPKMExperimental group/FPKMControl groupAnd (3) if the ratio of (a) to (b) is 2 or more, extracting a transcript unique to the experimental group relative to the control group as downstream analysis data. The invention effectively solves the problem of error assembly of the existing mRNA fragments, improves the credibility of differential analysis, realizes the quick search of the specific transcript of an experimental group, and enables the transcriptome data to further reflect the objective authenticity of a research object.
Description
Technical Field
The invention belongs to the technical field of RNA sequencing, and relates to a rapid detection method for mRNA5 '3' terminal difference.
Background
RNA sequencing and transcriptome analysis are powerful tools and methods for explaining the molecular mechanism of the life process, and provide basic and rich research and development data for life science industries such as downstream medicine, agriculture and the like by systematically analyzing an expression regulation network. The mRNA is an important object of transcriptome research, directly reflects the expression condition of active regions of a genome in existence or not and how much, can intuitively establish the corresponding relation between gene expression and phenotype of a life body by matching with comparative analysis among samples, and is beneficial to researching the basic process of life from the basic nucleic acid molecular level of life.
mRNA sequencing work mainly focuses on quantitative difference analysis, and two paths are provided before quantification to obtain the number of mRNA fragments (reads or fragments) for quantification, wherein one is to perform difference analysis by comparing an existing genome annotation file with a known sequence to obtain the existing sequence expression quantity of a database; secondly, based on the known mRNA fragment sequence, the real sequencing sequence (read) is combined for head assembly. The assembly result not only can be used for carrying out expression quantity difference analysis, but also can be used for finding out potential different transcripts (isofrorm) in corresponding experiments, and the specific transcripts are research targets of gene functions or life processes.
For mRNA fragment (read) assembly, Cufflinks and StringTie are two common software, the latter is an updated version of the former, and the StringTie improves the detection sensitivity of new transcripts and has shorter running time in addition to keeping the basic properties of the former. However, the improvement of the sensitivity brings about a plurality of incredible false positive assembly results, and causes pollution to objective single positive results; meanwhile, when the coverage of mRNA fragments (reads) is not high, the software still considers to be reliable and derives an assembly result, so that when a subsequent experimental group and a control group are compared in pairs, positive assembly fragments are erased, and a false negative result is caused.
Disclosure of Invention
The invention provides a simple, high-efficiency and high-precision rapid detection method for mRNA5 '3' terminal difference aiming at the errors introduced by the existing software in the process of de novo assembly of mRNA fragments (reads).
The technical scheme of the invention is as follows:
a method for rapidly detecting the difference of the 5 '3' terminal of mRNA comprises the following steps:
step 1, using HISAT2 to compare mRNA fragments (read) measured by a control group and an experimental group to a GRCh38 reference sequence, and selecting fragments of an intergenic region for subsequent analysis through sequence annotation information;
step 2, assembling the reads one by utilizing the overlapping sequence among the reads to form a5 'or 3' extended sequence of the gene;
step 3, forming an isoform (isoform) transcript based on the mRNA5 'or 3' elongation sequence of the gene, comparing the experimental group with the control group if the experimental group is longer than the control group by 200nt and FPKM is satisfiedExperimental group/FPKMControl groupIf the ratio of the two is more than or equal to 2, the difference between the tail ends of the experimental group and the control group is obvious, and then the specific transcript of the experimental group relative to the control group is extracted and used as downstream analysis data; if the length of the experimental group is not satisfied, the length of the control group is more than 200nt and FPKM is adoptedExperimental group/FPKMControl groupThe ratio of (a) to (b) is greater than or equal to 2, which indicates that the difference between the ends of the experimental group and the control group is not significant, and no subsequent analysis is performed.
In the embodiment of the present invention, FPKM is set for improving the credibility of the assembly sequencebinAnd FPKMextTwo parameters, FPKMbinRepresenting the read expression per bin unit (selected sequence window), FPKMextThe expression level of read representing the total length of extension, both of which are generally greater than 1, is reliable. Meanwhile, the window sequence length parameter bin can be manually set according to actual operation requirements, the smaller the numerical value is, the more accurate the assembly result is, but the more time is consumed. In a specific embodiment of the present invention, the window sequence length parameter bin is set to 10 nt.
Compared with the prior art, the invention has the following advantages:
the invention effectively solves the problem of error assembly of the existing mRNA fragments, improves the credibility of differential analysis, realizes the quick search of the specific transcript of an experimental group, and enables the transcriptome data to further reflect the objective authenticity of a research object.
Drawings
FIG. 1 is a schematic flow chart of the method for rapidly detecting the difference in 5 '3' terminal of mRNA according to the present invention.
Fig. 2 is a graph showing the results of comparison between the experimental group and the control group generated in the examples.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will also recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances.
Example 1
The flow of the rapid detection method for mRNA5 '3' end difference of the invention is shown in figure 1, and comprises the following steps:
(1) collecting single-stranded RNA fragments (reads) aligned to intergenic regions
Downloading a FASTA file of a GRCh38 genome sequence and a corresponding GTF annotation file, generating an exon and cut site file according to annotation contents by using HISAT2 software, establishing an index through the generated two files, and then completing alignment by using HISAT2 to generate a SAM file. Alignment procedure-k parameter was set to 1, i.e. only one of the corresponding RNA fragments in the best aligned position was retained.
(2) Elongation of fragment (read) Assembly determination of actual unique transcripts
From the alignment, a fragment (read) of the intergenic region was extracted. Setting bin Window Length to 10nt, FPKMbinAnd FPKMextBoth greater than 1, and then assembled stepwise with overlapping sequences between the fragments. After assembly, 5 'or 3' extension sequences are obtained. Comparing the experimental group with the control group, the length of the experimental group longer than the control group is set to be more than 200nt, and the FPKM is effectiveExperimental group/FPKMControl groupThe ratio of (a) to (b) is not less than 2, and finally, a transcript unique to the experimental group relative to the control group is extracted, and a result file is generated as follows and is shown in fig. 2.
Claims (3)
1. A method for rapidly detecting the difference of the 5 '3' terminal of mRNA is characterized by comprising the following steps:
step 1, comparing read measured by a control group and an experimental group to a GRCh38 reference sequence by using HISAT2, and selecting fragments of intergenic regions for subsequent analysis through sequence annotation information;
step 2, assembling the reads one by utilizing the overlapping sequence among the reads to form a5 'or 3' extended sequence of the gene;
step 3, forming an isoform transcript from the mRNA5 'or 3' elongation sequence of the gene, comparing the experimental group with the control group if the experimental group is longer than the control group by 200nt or more and the FPKM is satisfiedExperimental group/FPKMControl groupIf the ratio of the two is more than or equal to 2, the difference between the tail ends of the experimental group and the control group is obvious, and then the specific transcript of the experimental group relative to the control group is extracted and used as downstream analysis data; if the length of the experimental group is not satisfied, the length of the control group is more than 200nt and FPKM is adoptedExperimental group/FPKMControl groupThe ratio of (a) to (b) is greater than or equal to 2, which indicates that the difference between the ends of the experimental group and the control group is not significant, and no subsequent analysis is performed.
2. The rapid test method according to claim 1, wherein in step 1, FPKM is setbinAnd FPKMextTwo parameters, FPKMbinRepresenting the amount of read expression per bin unit, FPKMextRepresenting the amount of read expression extended over the total length, FPKMbinAnd FPKMextAre all greater than 1.
3. The rapid detection method according to claim 1, wherein in step 1, the window sequence length parameter bin is set to 10 nt.
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