RNA Sequencing
Description
RNA Sequencing, sometimes referred to as transcriptome sequencing, is used to reveal the presence, quantity and structure of RNA in a biological sample under specific conditions. Compared to hybridization-based RNA quantification methods such as microarray analysis, sequencing-based transcriptome detection can quantify gene expression with low background, high accuracy and high levels of reproducibility within a large dynamic range. In addition, transcriptome sequencing does not require an existing genome sequence and can detect splice variants and fusion genes that cannot be detected by microarrays.
Our experience, sequencing scale and proprietary technology enables quality RNA data, rapidly delivered, all at industry beating pricing. Contact us today for a no obligation quote.
Advantages
DNBSEQ (DNBSEQ Technology) platforms/technologies are utilized to deliver optimal results:
- Whole transcriptome profiling analysis of any species: the most comprehensive analysis of transcriptome of any species without specific probes and reference genomes.
- High coverage: digital signal and direct determination of almost all transcript fragments sequence.
- Wide detecting range: accurate quantification of a few to hundreds of thousands copies and identification of normal and rare transcripts.
- High resolution: detection of single base differences caused by alleles of gene families and alternative splicing.
- Digital signal: direct determination of every fragment sequence of transcripts, the single-base resolution, and no problems with cross reactions and background noise owing to cross-hybridization of the traditional tiling array.
Stranded Libraries Benefit
Stranded libraries have significant advantages over non-stranded libraries, including:
- Enabling detection of the antisense expression.
- More accurate quantification of overlapping transcripts.
- Higher-confidence discovery of alternative transcripts, gene fusions, and allele-specific expression.
- Enhanced confidence of transcript annotation.
- Increased mapping rate.
Key Service Details
- Reports and output data files are delivered in industry standard file formats: BAM, .xls, .png and FASTQ data.
Sequencing Standards
- Guaranteed ≥ 80% of bases with quality score of ≥ Q30
- 100bp and 150bp paired-end sequencing options available
- ≥30 million reads per sample recommended
Sample Requirements
Human/Mice/Rat (non-whole blood)
Total RNA: ≥ 200 ng,
Concentration: ≥ 10 ng/μl
General Quality Requirements: RIN: ≥ 7.0, 28S/18S: ≥ 1.0
Human Whole Blood
Total RNA: ≥ 500 ng,
Concentration: 40-1000 ng/μl
General Quality Requirements: RIN: ≥ 7.0, 28S/18S: ≥ 1.0
Animals, Except Human/Mice/Rat
Total RNA: ≥ 1 μg
Concentration: 40-2500 ng/μl
General Quality Requirements: RIN: ≥ 7.0, 28S/18S: ≥ 1.0
Plant
Total RNA: ≥ 1 μg
Concentration: 40-2500 ng/μl
General Quality Requirements: RIN: ≥ 6.0, 28S/18S: ≥ 1.0
Insect
Total RNA: ≥ 1 μg
Concentration: 40-2500 ng/μl
General Quality Requirements: RIN: ≥ 6.0, 28S/18S: ≥ 1.0
Fungi
Total RNA: ≥ 1μg.
Concentration: ≥ 40 ng/μl.
General Quality Requirements: RIN: ≥ 6.5, 28S/18S: ≥ 1.0.
Note: we can process degraded samples at lower quality thresholds. Contact us for more information.
Turnaround Time
- Typical 25 working days, from sample QC acceptance to filtered raw data availability.
- There is fast 18 working days TAT for urgent projects.
- Expedited services are available, contact us for more detail
FAQ
Q1: What is the difference between the stranded and the non-stranded mRNA library construction?
A1: The main differences between the stranded and the non-stranded mRNA library are listed below:
(1) In the cDNA 2nd strand synthesis step, use dUTP instead of dTTP.
(2) In the PCR step, prior to PCR amplification, the dUTP-marked strand is selectively degraded by UracilDNA-Glycosylase (UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing.
Q2: What are the differences in sample requirements, recommended data amount and analysis terms between the stranded and the non-stranded transcriptome sequencing?
A2: They are the same in these respects. Although, analysis terms are the same, the genome mapping and calculate gene expression level analysis parameters are a little different. Therefore, before bioinformatics analysis (including De novo and resequencing), the bioinformatics analysts need to pay attention to the method of library construction (stranded mRNA library or non-stranded), so that the correct analysis parameters can be set accordingly. And the base separation ratio of stranded RNA library is slightly higher. This is a characteristic analysis result of stranded RNA library.
Q3: What are the library types can offer for DNBSEQ (DNBSEQ Technology) transcriptome sequencing, and what is the differences between those library types?
A3: Different research aims target different library types. Different species, have different protocols/kits. Always remember: Good quality samples, good libraries and good sequencing result. For the standard library type is Stranded mRNA Library, polyA based mRNA enrichment, stranded library type. For other RNA library types, usually library types are PolyA based enrichment and rRNA depletion method.
