阅读说明：本技术 一种可高效用于黏细菌原核转录组建库测序的方法 (Method for efficiently sequencing viscobacteria prokaryotic transcriptome library ) 是由 董红红 朱红惠 董义杰 姚青 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种可高效用于黏细菌原核转录组建库测序的方法。它包括样品准备、RNA提取、mRNA富集及片段化,将mRNA反转录第一链cDNA,再合成第二链cDNA、构建文库、上机测序和对数据进行分析,其特征在于,所述的将mRNA反转录第一链cDNA,其使用的逆转录酶是Uni Reverse Transcriptase逆转录酶。本发明通过实验的手段证明了利用耐高温的逆转录酶可以显著提升转录的效率,有效提升高GC含量细菌的原核转录组测序成功率。(The invention discloses a method for efficiently sequencing a myxobacteria prokaryotic transcriptome library. The method comprises the steps of sample preparation, RNA extraction, mRNA enrichment and fragmentation, reverse transcription of mRNA into first strand cDNA, synthesis of second strand cDNA, library construction, sequencing on a computer and data analysis, and is characterized in that the mRNA is reverse transcribed into the first strand cDNA, and reverse transcriptase is used)

1. A method for efficiently sequencing a myxobacteria prokaryotic transcriptome library comprises the steps of sample preparation, RNA extraction, mRNA enrichment and fragmentation, reverse transcription of mRNA into first strand cDNA, synthesis of second strand cDNA, library construction, sequencing on a computer and data analysis, and is characterized in that the mRNA is reverse transcribed into the first strand cDNA, and reverse transcriptase is usedUni Reverse Transcriptase.

2. The method of claim 1, wherein the reaction system for reverse transcribing mRNA into first strand cDNA is: 500ng of the fragmented mRNA was obtained,uni Reverse Transcriptase Enzyme Mix 1 μ L,0.1 μ g/μ L Random Primer 1 μ L, 2 XTS-Uni Reaction Mix 10 μ L, RNase-free Water up to 20 μ L; the final reaction condition is that after incubation for 10min at 25 ℃, incubation for 30min at 52 ℃ is carried out, and the reaction is finished after cooling to 10 ℃.

3. The method of claim 2, wherein the reverse transcription of the mRNA into first strand cDNA is performed by mixing the fragmented mRNA, RNase-free Water and Random Primer, reacting at 65 ℃ for 5min, then cooling in ice for 2min, and adding 2 XTS-Unireaction Mix andand (3) incubating the Uni Reverse Transcriptase Enzyme Mix at 25 ℃ for 10min, incubating at 52 ℃ for 30min, and cooling to 10 ℃ to finish the reaction.

The technical field is as follows:

the invention belongs to the fields of molecular biology and high-throughput sequencing, and particularly relates to a sequencing method for a slime bacteria prokaryotic transcriptome library.

Background art:

slime bacteria are a group of bacteria widely distributed in soil and having predation characteristics, and most slime bacteria can prey on fungi, bacteria, small algae, protozoa and the like in soil through wolf-pack population movement behaviors. In addition, slime bacteria, which are predators located at the top of the microbial soil food chain and can regulate soil microecological balance through predation, are considered as a new microbial group with multiple biocontrol properties.

It is generally believed that in the presence of prey, cells of slime bacteria move in a coordinated manner to form swarms, which, when brought into contact with prey, can invade the flora and kill and lyse prey cells as a weapon of attack by secreting extracellular lytic factors, such as antibacterial substances, secondary metabolites, extracellular enzymes, etc., and can utilize the proteins released after lysis of prey cells as nutrients for self-growth. Due to the high complexity of the predation behavior of the slime bacteria, the research on the predation of the slime bacteria mainly focuses on the description of behavior characteristics, and the determined predation factors of the slime bacteria are only the aforementioned myxomycin A and the outer membrane type hydrolase GluM, and the predation factors of the slime bacteria which mediate cell lysis in different predation systems are not completely identified, but the identification of the predation factors of the slime bacteria in a specific predation system and the analysis of the action mode of the predation factors are the key for analyzing the predation mechanism of the slime bacteria.

It is possible to identify some extracellular lytic factors involved in predation by omics analysis. The transcriptomic analysis means is adopted to discuss the gene spectrum which is started or improved by the predator responding to the prey at the omic level and the prey enzyme spectrum secreted by the predator, so that all transcript information of the specific microorganism in a specific state can be comprehensively and quickly obtained, the expression quantity and the differential expression gene of mRNA and the corresponding function thereof are analyzed, and the molecular regulation mechanism and the function formed by different phenotypes of the microorganism are disclosed.

So far, there are only 5 reports on research on development process and predation mechanism of myxobacteria by using sequencing means of prokaryotic transcriptome, and only 1 report on transcriptomics research after the myxobacteria prey on the bacteria. And an effective prokaryotic transcriptome library-building sequencing method for a mixed sample after prey on prey bacteria by slime bacteria is not established at home and abroad.

The invention content is as follows:

in order to overcome the defects of the standard prokaryotic transcriptome sequencing technology widely adopted by various large biological companies, the invention solves the technical problem of providing a method for efficiently constructing a specific chain specificity cDNA library by a bacterial prokaryotic transcriptome with high GC content such as mucobacteria, can construct a high-quality sequencing library by using the method, ensures the high mapping rate (more than 90 percent) of sequencing off-machine data and a reference genome, has low cost and excellent effect compared with imported reagents by using high-temperature resistant reverse transcriptase, and provides a new technical method for carrying out the sequencing of the prokaryotic transcriptome with high GC content such as mucobacteria in the future.

According to the invention, the problem of low mapping rate of sequencing machine-off data and reference genome is found by experiments when the transcriptome sequencing is carried out on the slime bacteria by using the prokaryotic transcriptome sequencing standard flow of various domestic biological companies. The whole experimental process including sample preparation, RNA extraction, enucleated ribosome RNA enrichment mRNA and ncRNA, strand specificity library construction, on-machine sequencing and the like is analyzed, and the pollution does not exist in the sample preparation and RNA extraction processes and the pollution does not exist in the sequencing process. It is therefore speculated that the available libraries may not be suitable for high GC content bacteria such as slime bacteria.

Coli strain containing a plasmid cloned with M-MuLV RT gene, from which RNAase H core was deleted. M-MuLV Rtase is an RNA-dependent DNA polymerase which can synthesize a complementary DNA strand lacking RNAse H activity using a single-stranded RNA or DNA as a template in the presence of a primer. The MMuLV is mainly characterized in that the RNase H activity is ultra-low, the cDNA yield is high, and the MMuLV is a reverse transcriptase widely used in the prokaryotic transcription library construction process of various domestic large biological companies.

The method for efficiently sequencing the slime bacteria prokaryotic transcriptome library comprises the steps of sample preparation and RNA extractionmRNA is enriched and fragmented, mRNA is reversely transcribed into first strand cDNA, then second strand cDNA is synthesized, a library is constructed, and the data are analyzed through computer sequencing; the reverse transcription of mRNA into first strand cDNA using reverse transcriptase isA Uni Reverse Transcriptase,

in the present invention, theThe Unireverse Transcriptase Reverse Transcriptase is a high-temperature resistant Reverse Transcriptase produced by Beijing all-style gold biotechnology limited, and compared with the high-temperature resistant Reverse Transcriptase imported from abroad, the use of the enzyme can successfully realize the construction of a chain specificity library of a prokaryote with high GC content, such as myxobacteria, and can greatly reduce the experiment cost.

Further preferably, the optimal reaction system for first strand cDNA synthesis is: 500ng of the fragmented mRNA was obtained,uni Reverse Transcriptase Enzyme Mix 1 μ L,0.1 μ g/μ L Random Primer 1 μ L, 2 XTS-Uni Reaction Mix 10 μ L, RNase-free Water up to 20 μ L; the final reaction condition is that after incubation for 10min at 25 ℃, incubation for 30min at 52 ℃ is carried out, and the reaction is finished after cooling to 10 ℃.

Preferably, in order to increase the reverse transcription efficiency, the fragmented mRNA, RNase-free Water and Random Primer are mixed, reacted at 65 ℃ for 5min and then ice-washed for 2min, and then 2 XTS-Uni Reaction Mix andand (3) incubating the Uni Reverse Transcriptase Enzyme Mix at 25 ℃ for 10min, incubating at 52 ℃ for 30min, and cooling to 10 ℃ to finish the reaction.

The method can be used for constructing the chain specificity library of the sample after the slime bacteria prey on different prey bacteria, and improves the accuracy and reliability of the sequencing result of the prokaryotic transcriptome. The invention aims to realize the successful transcriptomic sequencing analysis of prokaryotes with high GC content, such as slime bacteria, and lays a foundation for researching the predation mechanism of slime bacteria from the omic level.

Compared with the prior art, the invention has the advantages that:

the invention proves that the transcription efficiency can be obviously improved by utilizing the high-temperature resistant reverse transcriptase through experimental means, and the sequencing success rate of the prokaryotic transcriptome of the bacterium with high GC content is effectively improved. Aiming at the current situation that the mapping rate of a novel microorganism group with predatory characteristics such as mucobacteria is low in the process of transcriptome sequencing, a technical method capable of carrying out transcriptomics sequencing research on samples of the mucobacteria and prey bacteria thereof is established by exploring and optimizing reverse transcriptase and reverse transcription temperature in the process of constructing a chain specificity library through a large number of pre-experiments.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention. The invention is further described with reference to specific embodiments. It should be noted that the following examples are intended to further illustrate the present invention, but are not intended to limit the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1:

the method for sequencing the slime bacteria prokaryotic transcriptome library comprises the following steps:

1. sample collection

Collected on TPM oligotrophic plates (MgSO)₄·7H₂O 1.97g/L，1M Tris-HCl pH 7.6 10mL/L，1MK₂HPO₄1mL/L, agar 15g/L, pH 7.2) of the individual growth of slime bacteria M.xanthothus e-3-1 and samples of tomato ralstonia r.solanacearum RS04 and e-3-1 prey RS04 (only samples at the prey boundary were collected), liquid nitrogen quick frozen and placed in a-80 ℃ freezer for subsequent experiments.

2. Extraction and quality control of sample RNA

RNA is extracted from the collected thalli by a standard Trizol method, and then the RNA sample is strictly controlled by an Agilent 2100bioanalyzer, and the RNA integrity and the total RNA amount are accurately detected.

3. Enrichment and fragmentation of mRNA

After the extracted RNA is detected to be qualified, the ribosome RNA (rRNA) in the Total RNA is removed to obtain the mRNA.

4. Building warehouse

The method comprises the following steps of establishing a library by using a standard prokaryotic transcriptome library establishing program of Beijing Nuo Zhiyuan Biotechnology GmbH:

first strand cDNA synthesis: first strand cDNA is synthesized in M-MuLV reverse transcriptase system by using fragmented mRNA as template and random oligonucleotide as primerUnireverse Transcriptase replaces M-MuLV Reverse Transcriptase to serve as an experimental group), RNA chain is degraded by RNaseH, second chain cDNA is synthesized by taking dNTPs as raw materials under a DNA polymerase I system, the end of the purified cDNA is repaired, A tail is added, a sequencing joint is connected, AMPure XP beads are used for screening cDNA about 370-420bp, the USER enzyme is used for degrading the second chain cDNA containing U, PCR amplification is carried out, PCR products are purified by the AMPure XP beads again, and finally a library is obtained.

The first strand cDNA is synthesized (usingUni Reverse Transcriptase) is as follows: 500ng of the fragmented mRNA was obtained,1 μ L of Uni Reverse Transcriptase Enzyme Mix, 1 μ L of 0.1 μ g/μ LRandom Primer, 10 μ L of 2 XTS-Uni Reaction Mix, and 20 μ L of RNase-free Water up to; the final reaction condition is that after incubation for 10min at 25 ℃, incubation for 30min at 52 ℃ is carried out, and the reaction is finished after cooling to 10 ℃. Specifically, the fragmented mRNA, RNase-free Water andmixing Random primers, reacting at 65 deg.C for 5min, ice-cooling for 2min, adding 2 XTS-Uni Reaction Mix andand (3) incubating the Uni Reverse Transcriptase Enzyme Mix at 25 ℃ for 10min, incubating at 52 ℃ for 30min, and cooling to 10 ℃ to finish the reaction.

After the library is constructed, firstly, a Qubit2.0 Fluorometer is used for preliminary quantification, the library is diluted to 1.5ng/ul, then, an insert size of the library is detected by using an Agilent 2100bioanalyzer, and after the insert size meets the expectation, qRT-PCR is used for accurately quantifying the effective concentration of the library (the effective concentration of the library is higher than 2nM) so as to ensure the quality of the library.

5. Sequencing on machine

And after the library is qualified, performing Illumina sequencing on different libraries according to the effective concentration and the requirement of the target offline data volume.

6. Sequencing off-line data quality control

The image data of the sequencing fragment obtained by a high-throughput sequencer is converted into sequence data (reads) through CASAVA base recognition, the reads containing N (N represents that base information cannot be determined) and the low-quality reads (reads with the base number of Qphred < (20) > accounting for more than 50% of the length of the whole read) to be spliced are removed, the original sequencing sequence obtained by sequencing is filtered to obtain clearreads, and the sequencing error rate distribution and the GC content are further analyzed.

7. Reference genomic sequence alignment analysis

The filtered sequences were subjected to genomic mapping analysis using Bowtie2(Langmead, b.2012) software.

Table 1 shows the comparison statistics of the sample and the reference genome after library construction and sequencing of samples before and after the tomato Ralstonia solanacearum RS04 predation by the Myxococcus xanthus e-3-1, a mucor, by using a standard prokaryotic transcriptome library construction and sequencing process of Beijing Nonghe biogenic biotechnology Co.

Table 2 shows the first chain c for synthesis in the chain specificity library construction in the sequencing process of the standard prokaryotic transcriptome library construction of the biogenetic biotechnology of Beijing Nuo HeiyuanM-MuLV reverse transcriptase of DNA is replaced by the high-temperature resistant reverse transcriptase of the inventionAfter the Uni Reverse transcription, other samples before and after the sample library construction and sequencing of the mucoid Myxococcus xanthus e-3-1 of the tomato Ralstonia solanacearum RS04 are compared with the reference genome for statistics in the same original standard process.

In the absence of contamination in the relevant experiments, the method is usedThe Uni Reverse Transcriptase synthesizes first strand cDNA to construct a strand specific library, sequencing Reads generated by experiments are successfully compared until the proportion of genomes is higher than 90% (Total Mapped Reads or Fragments), and the problem of low mapping rate when the traditional method is used for constructing the strand specific library to carry out transcriptome sequencing analysis on bacteria with high GC content such as mucobacteria is solved.

TABLE 1 statistics of sample to reference genome alignments

TABLE 2 statistics of sample to reference genome alignments

Total reads: quantitative statistics of the sequenced sequence after filtering of the sequencing data (Clean data)

Total mapped: statistics of the number of sequencing sequences that can be mapped onto the genome; in general, the percentage of this portion of data is greater than 70% if contamination is not present and the reference genome selection is appropriate

Multiple mapped: a statistical number of sequenced sequences having a plurality of aligned positions on the reference sequence; the percentage of this portion of data will generally be less than 10%

Uniquely mapped: statistical quantification of sequenced sequences with unique alignment positions on the reference sequence

Read-1: counting the number of the sequenced sequence Read-1 aligned to the genome, and the meaning of Read-2 is similar to that of Read-1

positive _ map, negative _ map: statistics of sequencing sequence alignment to Positive and negative strands on the genome

Reads mapped in paper documents, paper-printed Reads map to differential chrome: statistics of pairwise sequencing sequence alignment to reference genome and pairwise sequencing sequence alignment to different chromosomes.