(Second Generation High Throughput Sequencing Technology - 454) Second, the outstanding advantages of Meiji Company in the study of environmental microbial biodiversity are reflected in: Third, the service process (1) The customer provides sample background information, experimental purposes and experimental expectations. IV. Sample delivery requirements (1) Animals, plants, microbial tissues: V. No reference genome transcriptome analysis (de novo Transcriptome Analysis) Sixth, transcriptome analysis with reference genome (Transcriptome analysis with reference genome) VIII. Frequently Asked Questions (1) Is it possible to perform prokaryotic transcriptome analysis? Accompany Chair,Recliner Chair Price,Accompany Recliner Chair,Medical Accompany Chair Jiangyin Kaili Health-Care Equipment CO., LTD. , https://www.kailimed.com
The transcriptome, the sum of all the RNAs that a particular cell can transcribe under a certain functional state, is an important means of studying cell phenotype and function. Unlike the genome, the definition of the transcriptome contains time and space constraints. The gene expression of the same cell in different growth stages and growth environments is not completely the same. The Roche GS-FLX-Titanium second-generation high-throughput sequencer has an average read length of more than 400 bp and is far ahead of other second-generation high-throughput sequencers in sequencing, making it the preferred sequencing platform for transcriptomics research. Has been widely used in basic research, clinical diagnosis and drug development.
First, the outstanding advantages of Roche 454 sequencing technology in the study of environmental microbial biodiversity are reflected in:
(1) The sequencing sequence is long, which facilitates clustering and splicing, and de novo assembly can be performed on the transcript.
(2) High sequencing flux can detect low abundance transcript information.
(3) Transcriptome sequencing of new species without a genome-based reference sequence can be performed to discover new transcripts and subtypes.
(4) The experiment is simple, the result is stable, and the repeatability is strong. The library construction without cloning is not required, and the double-stranded cDNA can be directly sequenced after the 454 linker, and the experimental period is short.
(5) Sequencing data facilitates bioinformatics analysis, enabling differential gene expression analysis, identifying variable splicing of genes, and predicting new genes.
(1) With independent laboratory and high-throughput sequencing platform, the experiment can be arranged flexibly according to customer requirements, with short experimental period, convenient sampling and reliable quality.
(2) The experienced technicians can stably extract total RNA and synthesize double-stranded cDNA, and can provide experimental protocols at the first time according to customer requirements.
(3) A professional bioinformatics team and a large computer can provide customers with personalized bioinformatics analysis services.
(4) Open laboratory, participatory services. Customers can not only participate in the entire experimental process, but also participate in bioinformatics analysis to provide the most value-added after-sales service.
(2) Miki designed the experimental protocol to provide recommendations for sequencing depth and bioinformatics analysis.
(3) The client approves the experimental plan and the two parties sign a project cooperation agreement.
(4) The project started to operate, and Meiji Company designated special personnel and customers to maintain barrier-free communication.
(5) At the end of the project, Meiji provides a standard completion report.
(6) Customers can sign a long-term cooperation agreement with Meiji Company to enjoy discounts and VIP services.
> Please provide a sufficient amount of fresh samples, the sample size ≥ 5g; plant materials should avoid the old tissue, try to use soft parts.
> Freshness requirement: Immediately after sampling, the sample will be frozen at -80 °C (with a shelf life of less than 1 month), transported on dry ice, and transported for no more than 72 hours.
> Avoid freezing and thawing during sample storage.
> Judging criteria for the quality of samples: After RNA extraction, the RNA bands are clear, the ratio is moderate, the integrity is good, and there is no degradation.
> Complete the sample delivery order (please click download) to provide as much detailed information as possible on the background of the species genome, such as: genome size, average gene length, and the number of genes expected to be expressed. Seal with a ziplock bag and send it with the same copy.
> For sample delivery and other specific requirements, please call for free.
(2) RNA
> total RNA>500ug, concentration >1ug/ul;
> mRNA>3ug, concentration>50ng/ul;
> OD260/OD280 is 1.8-2.2; 28S/18S>1.8; RIN≥ 8 (mammal)
> The quality inspection is based on our electrophoresis gel and UV analyzer. .
> The sample tube must be marked with the sample number and the nozzle is sealed with a Parafilm membrane.
> Avoid freezing and thawing during sample storage.
> Use dry ice for transport when delivering samples.
> Complete the sample delivery order (please click to download) and RNA electrophoresis test photos, seal with a ziplock bag and then deliver with the same.
(3) Double-stranded cDNA
> Library construction was performed according to the cDNA 454 library construction procedure provided by Miki, and the POLY-A in the library was removed.
> The total amount is >5ug, the concentration is >50ng/ul, and the OD260/280 is around 1.8.
> The sample tube must be marked with the sample number and the nozzle is sealed with a Parafilm membrane.
> Avoid freezing and thawing during sample storage.
> Use dry ice or ice packs for sample delivery.
> Complete the sample delivery order (please click to download) and the cDNA electrophoresis test photo, seal it with a ziplock bag and send it with the same copy.
> Sequencing and basic data processing Synthesis of double-stranded Cdna enzymes cleavage of POLY-A 454 linker at both ends 454 Sequencing Basic data processing (image recognition, base recognition, filter linker sequence)
> Bioinformatics analysis content
* Composition and quality assessment of data output statistics and sequencing data
* Contig length distribution
* Unigene length distribution
* Unigene function annotation
* Unigene GO classification
* Unigene expression difference analysis (not less than two samples)
* Unigene's difference in sample GO classification (not less than two samples)
* Unigene metabolic pathway analysis
* Protein function and classification prediction
> Application
* Whole genome EST sequencing
* Screening for SNV, SSR and InDel molecular markers
* Gene expression profiling
* Non-coding RNA research
> Sequencing and basic data processing Synthesis of double-stranded Cdna enzymes cleavage of POLY-A 454 linker at both ends 454 Sequencing Basic data processing (image recognition, base recognition, filter linker sequence, detection of possible sample contamination)
> Bioinformatics analysis content
* Sequencing data yield and overview of comparison results with Reference
* Distribution of Reads on the genome
* Sequencing randomization assessment
* Distribution of gene coverage and sequencing depth
* Gene differential expression analysis
* Optimize gene structure
* Identification of variable shear of genes
* Predict new genes
> Application
* Genome annotation
* Gene structure analysis (variable splicing, gene boundaries, etc.)
* Transcriptional fusion gene research
* Gene expression profiling
* Non-coding RNA research
> Yes. Please provide 20 ug total RNA samples.
(2) What samples do I need to provide?
> Can provide fresh animals, plants, microbial tissues, extracted total RNA or synthetic double-stranded cDNA.
(3) How long is the experiment period?
> From the acquisition of customer samples, high-throughput sequencing to data analysis, the company's individual control needs to be completed within 30 to 40 business days depending on the individual needs of the partner.
(4) How much sequencing is needed?
> 1 million effective sequences, with an average read length of 300 to 500 bp. After clustering, 2 to 40,000 Unigenes can be obtained. The average size of Unigene is between 500 bp and 10 Kb. The size of the transcriptome is affected by both the number of genes and the abundance of genes, and varies greatly from species to species. In addition, factors such as tissue differences, status, and experimental processing also affect the composition of the transcriptome.
(5) Can I participate in experiments and bioinformatics analysis?
> We are an open laboratory and we welcome partners to participate in our experimental process and data analysis process to enhance communication in participation.
(6) Judgment of full-length cDNA
> Whether the Unigene clustered and spliced ​​is a full-length cDNA needs to be judged.
> Directly from the sequence can be judged from the following aspects.
5' end:
* Comparison of homologous full-length genes is judged by Blast with the corresponding gene ends of other organisms.
* New genes without homologous genes, (1) to determine whether the coding framework is complete, a. There is a stop code for the same frame upstream of the first ATG in the open reading frame. It should be noted that sometimes the true translation initiation codon is not It is the first ATG that appears in the mRNA. In some eukaryotic cells, one to several ATGs may appear in the upstream non-coding region of the initiation codon ATG, which is called a non-coding 5' ATG. In this case, the 5' ATG is not a true start codon, and the open reading frame from which it begins often encounters a stop codon. b. If there is no stop code, consider a conservative Kozak sequence; (2) Determine whether it is from the transcription start point, there is data indicating that there is generally a region rich in pyrimidine after the 5' cap structure, and if the cDNA 5' sequence is The portion of the genomic sequence that is protected by S1 digestion is the same, and it can be confirmed that the obtained cDNA is full-length.
3' end:
* There is a comparison of homologous full-length genes, the same method as the 5' end;
* There is a termination password downstream of the coding framework;
* There is more than one polyA tail signal;
* There is also a polyA tail if there is no obvious tailing signal.
> Comparison of homologous full-length genes can be achieved using Blast alignment or multiple sequence alignment software. First search for other species (higher similarity species), full-length cDNA sequences, and then make these sequences Blast or multiple alignments.
> Determine the cDNA sequence to be a full-length cDNA sequence. It is only a "virtual clone" on a computer. Finally, it must be verified by RT-RCR, sequencing and Northern hybridization to ensure the accuracy of the sequence.