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RNA-SEQ (Transcriptome)

Overview

Compared to traditional array-based gene expression methods, RNA Sequencing (transcriptome sequencing) provides unique information about isoforms, splice variants and fusion genes that cannot be readily obtained using microarrays. In addition, RNA Seq transcriptome data provides a much larger dynamic range for high-resolution analysis of low- abundant transcripts. These advantages allow for more comprehensive understanding of the underlying biology at the RNA level. By running a high-throughput sequencing facility, BGI supports large-scale complex transcriptome sequencing, with an output ranging from 4Gb over 20Gb per sample, for large and complex projects.

BGI pioneers the use of long inserts coupled with long reads for transcriptome using the Illumina HiSeq 4000 system. The benefits include high yields in transcripts, fusion genes and splice variants.In addition, our CAP and CLIA-compliant laboratories located in Hong Kong and the US, respectively, provide exceptional data quality at very competitive cost levels. For clients who focus on drug development and clinical trial studies, BGI provides certified processes to comply with electronic signature and audit trail requirements specified by 21 CFR Part 11.

Service Offerings & Specifications

Industry-leading Quality

  • CAP-grade data for research and clinical applications
  • Client data from BGI’s CAP-compliant sequencing and data production environment
  • US FDA 21 CFR Part 11 compliant process

Unmatched Customer Support

  • 24/7 Worldwide technical support by NGS technical specialists and bioinformaticians
  • Secured, web-based customer report access
  • Company-wide commitment to zero-error sample handling logistics*

Sequencing Options at Variable Output Levels

  • 4Gb-20Gb or higher – BGI recommends 6Gb to 8Gb output for optimal power to detect minor transcript species
  • Raw-data only sequencing services available

Sequencing Platforms

  • Latest Illumina HiSeq 4000 system with 50, 75, 100 and 150bp paired-end and single end sequencing
  • Complete Genomics sequencing platform

Transcriptome Sequencing Data Analysis

  • Quantitative expression profiles
  • Spliced transcript analysis
  • Fusion gene analysis
  • SNP list
  • Indel list
  • RNA editing analysis
  • Available gene ontology analysis
  • Available hierarchical clustering

Turnaround Time

  • Standard turnaround time is 1-2 months
  • Rapid turnaround available

Data Security

  • ISO/IEC 27001:2005 Information Security Management System Certificate
  • All client data stored within BGI firewall protection paradigm

Customer Testimonials

“As the principal investigator on two sequencing projects that are pushing the state-of-the-art (viral discovery using metagenomic sequencing and phylogenomic analysis of 1000 plant species) I cannot expect everything to work on the first iteration but I do expect my sequencing provider to work together with me to correct whatever went wrong regardless of whose fault it was. BGI does that admirably.” Dr. Gane Ka-Shu Wong, Professor and iCORE Chair in Biosystems Informatics Department of Biological Sciences – University of Alberta

Technical Information

Bioinformatics:

BGI Tech provides two types of bioinformatics analyses: de novo and transcriptome resequencing.

De novo Transcriptome Assembly

  • Standard bioinformatics analysis
    1. Data filtering includes removal of adaptors and low-quality reads from raw reads
    2. Statistics analysis and evaluation of data
    3. Assembly results (Contig length distribution, Unigene* length distribution)
    4. Unigene function annotation and COG classification
    5. Unigene GO classification
    6. Unigene pathway analysis
    7. Coding region sequence (CDS) prediction
    8. Analysis of differential unigene expression (two or more samples should be provided)
    9. Gene ontology (GO) classification and pathway enrichment analysis of differentially expressed unigenes (two or more samples should be provided)
    10. SNP analysis
    11. SSR marker identification and primer design
  • Advanced bioinformatics analysis
    1. Principal component analysis (PCA) (five or more samples should be provided)
    2. Condition–specific expression analysis (five or more samples should be provided) (eukaryotes only)

Transcriptome Resequencing

  • Standard bioinformatics analysis (reference genes, genome sequences, and gene annotation should be provided)
    1. Data filtering includes removal of adaptors, contamination, and low-quality reads from raw reads
    2. Assessment of sequencing (alignment statistics, randomness assessment of sequencing, and distribution of reads along the reference gene)
    3. Gene expression and annotation (gene coverage and coverage depth)
    4. Analysis of differential gene expression (two or more samples should be provided)
    5. Expression pattern analysis of differentially expressed genes (DEGs)
    6. Gene ontology analysis of DEGs
    7. Pathway enrichment analysis of DEGs
    8. Refinement of gene structures (eukaryotes only)
    9. Identification of alternative spliced transcripts (eukaryotes only)
    10. Prediction and annotation of novel transcripts
    11. SNP analysis
  • Advanced bioinformatics analysis (eukaryotes only)
    1. Gene fusion analysis (for human only, preferably more than 15 pairs of control + case)
    2. Principal component analysis (PCA) (five or more samples)
    3. Condition–specific expression analysis (five or more samples)
    4. Correlation analysis between samples
    5. Venn diagram of gene distribution specific to one sample and/or across all test samples
    6. Visualization of genome alignment results using IGV
    7. Protein-protein interaction network analysis
    8. Transcription factor analysis (plant only)
    9. KEGG pathway enrichment scatter diagram
    10. Transcript quantification
    11. Exon quantification
    12. InDel analysis
    13. RNA editing analysis

Duplex-Specific Nuclease (DSN) Normalization Transcriptome Sequencing Bioinformatics Analysis

  • The analysis content is the same as that performed in de novo transcriptome sequencing except for the analysis of differentially expressed genes

Custom Bioinformatics Analysis

  • We can also perform other customized analyses to meet the requirements of specific projects.

Sample Requirements:

Regular transcriptome sequencing

  • Sample conditions: Total RNA samples that have been treated with DNase. Avoid protein contamination during RNA isolation.
  • Sample quantity (for single library construction):
    1. Pant and fungi: total RNA ≥ 20μg
    2. Bacteria: total RNA ≥ 5μg
    3. Mammal (human, rat and mouse): total RNA ≥ 5μg
    4. Other species: total RNA ≥ 10μg
  • Sample concentration:
    1. Plant and fungi: ≥ 250ng/µl
    2. Bacteria: ≥ 65ng/µL
    3. Mammal (human, rat and mouse): ≥ 65ng/uL
    4. Other samples: concentration ≥ 150ng/µl
  • Sample purity:OD260/280 = 1.8-2.2, OD260/230 ≥ 2.0
    1. Plant and fungi: 28S:18S RNA ≥ 1.0, RIN ≥ 6.5
    2. Bacteria: 23S:16S RNA ≥ 1.0, RIN ≥ 7.0
    3. Animal: 28S:18S RNA ≥ 1.0, RIN ≥ 7.0

Low-input transcriptome sequencing

Total RNA isolated from tumors or dead tissue can often be in limited amounts and poor quality. However, some of these sources are of clinical importance. To consistently achieve high-quality data from these types of samples, we recommend low-input transcriptome sequencing.

  • Sample conditions: Total RNA samples that have been treated with DNase. Avoid protein contamination during RNA isolation.
  • Sample quantity (for single library construction):
    1. Pant and fungi: total RNA ≥ 2μg
    2. Mammal (human, rat and mouse): total RNA ≥ 200ng
    3. Other species: total RNA ≥ 1μg
  • Sample concentration:
    1. Plant and fungi: ≥ 250ng/µl
    2. Mammal (human, rat and mouse): ≥ 65ng/uL
    3. Other samples: concentration ≥ 150ng/µl
  • Sample purity:OD260/280 = 1.8-2.2, OD260/230 ≥ 2.0
    1. Plant and fungi: RNA 28S:18S ≥ 1.0, RIN ≥ 6.5
    2. Animal: RNA 28S:18S ≥ 1.0, RIN ≥ 7.0

Turnaround Time:

The standard turnaround time for the workflow (above) is 40 business days.

Cases

The 1000 Plants de novo Transcriptomes Project

The 1000 plants de novo Transcriptomes Project plans to use new generation technology to de novo sequence and assemble the transcripts of 1,000 plants. This is an initiative project funded by the government of Alberta. BGI is one of the major participants of the 1000 Plants Initiative. BGI initially seeks to increase the number of plant species for which transcript sequence information is publicly available and to learn about their biology and evolutionary history. In later phases, the initiative might focus on commercial applications of the results. Fewer than 100 plant genomes have been characterized by sequencing so far, even at the EST level, judged by data submitted to GenBank. This project will greatly expand the knowledge of plant biodiversity.

Deep RNA Sequencing at Single Base-Pair Resolution Reveals High Complexity of the Rice Transcriptome. Genome Research 2010:646-654.

Deep-RNARNA sequencing enabled the detection of transcripts expressed at an extremely low level. The results suggest that transcriptional regulation in rice is vastly more complex than previously believed.

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  •   Whole Genome Resequencing
      Exome Sequencing
      Target Region Sequencing
      de novo Sequencing
      RNA-Seq (Transcriptome)
      RNA-Seq (Quantification)
      Small RNA Sequencing
      Long Non-coding RNA Sequencing
      Whole Genome Bilsulfite Sequencing
      Reduced Representation Bilsulfite Sequencing
      MeDIP Sequencing
      ChIP-Seq
      16S rDNA tagging
      Wole Genome Metagenomic Sequencing
      Microbial Gene Catalog
      Proteomics Services
      Others
  •   Human
      Others
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