Eukaryotic De Novo mRNA Sequencing

De novo sequencing directly sequences the genome or transcriptome without any sequence information as a reference, and use bioinformatics for sequence assembly and annotation. Therefore, when the species under study does not have a reference genome, through the de novo analysis strategy, mRNA sequencing can help researchers to quickly obtain the mRNA sequence, analyze gene expression levels, and more.

Overview

At present, there are still a large number of species without reference genome sequence. For these species, the basic information of their transcriptomes must be obtained through de novo transcriptome splicing. De novo mRNA sequencing splices and assembles the sequences through bioinformatics methods, which provides the information for gene functional annotation, differential analysis, SNP analysis of transcripts.

CD Genomics's eukaryotic de novo sequencing utilizes the Illumina sequencing platform to sequence all mRNAs transcribed by a specific eukaryotic tissue or cell in a specific state, and spliced ​​out the longest transcript as unigene for subsequent analysis, which provides a powerful technical mean for studying transcriptional level changes in species without reference genomes. After transcript splicing and annotation based on our state-of-the-art sequencing platform and bioinformatics software, gene expression level and structural information are analyzed in an all-round way to maximize the research content of species without genome reference. The information analysis of our de novo sequencing service has undergone strict data quality control to ensure the reliability of data analysis, which is divided into expression level analysis and structure level analysis.

Features

Good integrity Experienced Scientist Team Strong Reliability Professional Bioinformatics
Low-abundance transcripts can be obtained for more complete transcriptome. Can provide a full set of professional services from experimental design, sample testing, data analysis, etc. High data volume ensures that transcripts of different abundances can be accurately quantified. Strong bioinformatic team provides conventional analysis and in-depth data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation; crosslinking reaction; PCR amplification.

Sequencing

3. Sequencing Platform

Hiseq X Ten, PE150; 6 Gb per sample

Data Analysis

4. Data Analysis

Preprocess and visualize results, and perform custom bioinformatics analysis.

Eukaryotic De Novo mRNA Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • De novo sequencing quality control analysis
  • De novo assembly, validation, and evaluation
  • Gene prediction
  • Annotation analysis: searching of homology in sequence database; CAZy analysis; GO annotation analysis; Pathway analysis; COG annotation
  • Homologous prediction
  • CDS prediction; SNP analysis; SSR analysis
  • Genomic collinearity analysis
  • Phylogenetic tree construction

Sample Requirements

Tissue sample: above 100 mg

Cell sample: cell amount: above 1×107

RNA sample: RNA quantity ≥ 5 μg; RNA purity: OD260/280 = 1.6~2.3; OD260/230 ≥ 1.5; RNA quality: 28S:18S ≥ 1.5 or RIN ≥ 7

Please make sure that the RNA is not significantly degraded.

Sample storage: Cell samples or fresh tissue pieces (cut into 5-10 mg pieces) can be treated with TRIzol or RNA protectant, frozen in liquid nitrogen, and stored at -80°C. RNA samples can be dissolved in ethanol or RNA-free ultrapure water, and stored at -80°C. Avoid repeated freezing and thawing during sample storage.

Shipping Method: The samples should be stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

References:

  1. Moreton J, Izquierdo A, Emes RD. Assembly, assessment, and availability of de novo generated eukaryotic transcriptomes. Frontiers in genetics. 2016 Jan 11;6.
  2. McLysaght A, Guerzoni D. New genes from non-coding sequence: the role of de novo protein-coding genes in eukaryotic evolutionary innovation. Philosophical Transactions of the Royal Society B: Biological Sciences. 2015 Sep 26;370(1678).
* For Research Use Only. Not for use in diagnostic procedures.


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