RNA-SEQ

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RNA-Seq is a method for transcriptome profiling that uses next generation sequencing technologies. RNA-Seq provides a comprehensive, quantitative, and unbiased view of RNA sequences within every sample.
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Microarray technology utilizes a pre-defined set of probes to capture and quantify specific RNA sequences. This means that microarrays are capable of detecting only a pre-selected set of transcripts. RNA-Seq relies on next generation sequencing technologies, enabling the identification and quantification of any RNA sequence in the sample.
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The number of reads required depends upon the genome size, the number of known genes, and transcripts. GENEWIZ recommends 10 million reads for small genomes (i.e. bacteria) and 30 million reads for large genomes (i.e. human, mouse). A larger number of reads will generate more sequencing data, which increases the chances of detecting genes that are expressed at lower levels. Please note: A sufficient amount of sequencing data is necessary to ensure the quality of downstream data analysis results; an insufficient number of reads may lead to generating results that cannot be analyzed.

For RNA-Seq experiments, GENEWIZ recommends a 1x50 bp single-read (SR) configuration for projects looking to analyze gene expression levels, and a 2x100 bp paired-end (PE) configuration for projects looking to identify novel isoforms, such as alternative spliced transcripts. A 2x100 bp PE configuration is also recommended for projects that required de novo transcriptome assembly.

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At GENEWIZ, we recommend starting with total RNA. We also accept ds-cDNA, mRNA, and cell cultures and pellets.

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GENEWIZ Project Management, a team of dedicated technical experts, is available to assist with experimental design and to answer any questions for RNA-Seq projects. Project Management is accessible via email or by phone at +1-908-222-0711 ext 1 from 8:00 a.m. – 6:00 p.m. (ET).

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GENEWIZ provides raw data as FASTQ files for all projects. GENEWIZ also has advanced bioinformatics capabilities to provide optional data analysis services, including:
  • Reference mapping and raw hit count measurements to identify expression levels of individual samples;
  • Differential gene expression analysis to provide a statistical comparison of expression levels between multiple samples;
  • Novel isoform identification to detect known and unknown isoforms, such as alternatively spliced transcripts.

In addition, GENEWIZ provides de novo transcriptome assembly for sequences that do not have a reference. 

If you need custom data analysis, please provide analysis requirements in the project description section of the RNA-Seq Quote Request Form.

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Please complete the GENEWIZ RNA-Seq Sample Submission Form, and send your samples on dry ice to: 
 

GENEWIZ  

Attn: NGS Laboratory  

115 Corporate Boulevard  

South Plainfield, NJ 07080  

USA  

International orders: Please inform shipping agents that your package is perishable. Please send GENEWIZ the tracking information for your package upon shipment. Because international shipments process through Customs, please retain a sufficient amount of your samples in the event you need to resubmit materials to GENEWIZ.

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Yes, GENEWIZ accepts ds-cDNA as a starting material for RNA-Seq projects. Please note, GENEWIZ is unable to provide full quality control when using ds-cDNA. We request at least 1 µg of ds-cDNA with a concentration of ≥50 ng/µl.

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Yes, GENEWIZ accepts mRNA as a starting material. However, we will be unable to do a quality check before beginning the library construction process. We request > 20 ng of mRNA to start. Samples should be eluted or re-suspended in nuclease-free water.
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The following specifications apply to total RNA samples:

  • Total amount: ≥ 5 µg 
  • Concentration: ≥ 80 ng/µl
  • OD260/280 Range: 1.8-2.2
  • Re-suspended in nuclease-free water
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Yes, GENEWIZ can sequence ready-to-load libraries. Please send at least 15 µl volume of the libraries at a concentration of 10 nM on ice.
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GENEWIZ generally uses a poly-A selection method to enrich RNA transcripts for eukaryotic samples. In some cases, it is more effective to deplete ribosomal RNA (rRNA) as an alternative method to poly-A selection. For example, total RNA from species with a large amount of RNA would benefit from using rRNA depletion. Furthermore, if you need to detect several types of non-coding elements, such as long and short ncRNAs, we recommend using the rRNA depletion method.

For prokaryotic samples, GENEWIZ depletes the rRNA for all projects.

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GENEWIZ guarantees delivery of the number of reads selected for your samples. As a guideline, the delivery guarantee is 120 million reads per lane on the Illumina HiSeq 2500 in rapid run mode and 250 million reads per lane in high output mode.

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GENEWIZ may be able to work with less starting material. Please let us know your project requirements in the project description section of the RNA-Seq Quote Request Form.
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GENEWIZ provides updates upon sample receipt and upon confirmation of quality control results. Subsequently, GENEWIZ provides weekly updates throughout the duration of your project.

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Raw hit count is acquired by counting the number of reads non-ambiguously aligned to a particular genomic feature, typically a gene or transcript.

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Client confidentiality and protection of Intellectual Property (IP) are of the utmost importance at GENEWIZ. Clients take confidence in the security and privacy of all projects completed with GENEWIZ. For more information, please reference the GENEWIZ Confidentiality Policy.

WHOLE GENOME SEQUENCING

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Genomes that have not been sequenced before must be assembled de novo following sequencing.
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De novo assembly can present a bioinformatics challenge, in particular to close any gaps remaining following sequence assembly.

During the assembly process, you end up with large DNA sequences known as contigs formed from overlapping sequence reads. Since next generation sequencing (NGS) works by sequencing fragmented nucleic acid, gaps will exist between contigs.

TARGETED RESEQUENCING

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Targeted resequencing is an application of next generation sequencing where you enrich regions of the genome that you are interested in sequencing.


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The size of full genomes can be very large. Targeted resequencing lets you remove areas that you are not interested in knowing more about so that your initial target size is smaller. Since the output remains constant for a particular NGS platform and configuration, a smaller target size gives you the flexibility of multiplexing more samples into the same run or sequencing at a higher depth of coverage.

Increased ability to multiplex makes the project more cost-effective since you are sequencing more samples in a single run (significantly decreasing the per sample cost).

Higher depth of coverage gives the assay more sensitivity. This higher sensitivity allows you to detect low-frequency, rare mutations more effectively. Also, it allows for resolving more complex (e.g., GC- or AT-rich) genomic regions.

Exome sequencing is a form of targeted resequencing. However, the target size is significantly larger than most custom targeted assays. For this reason, the same benefits apply as compared to whole genome sequencing.

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There are primarily two types of targeted resequencing: targeted enrichment and amplicon sequencing. Targeted enrichment captures regions of interests with baits following creation of the full genomic library. By contrast, amplicon sequencing amplifies the target regions directly from the unfragmented genomic DNA in a highly multiplexed PCR reaction. Further, there are multiple targeted enrichment and amplicon sequencing technologies designed by different companies, including Illumina, Agilent, and Life Technologies.

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The assay design process can range from relatively straightforward to extremely complex.

Some key elements of the design process include:

  • Deciding between amplicon sequencing and targeted enrichment
  • Deciding on the best amplicon or enrichment chemistry/technology
  • Determining the best design for the particular regions/genes (e.g., a GC-rich or complex set of regions would be handled differently than a simple one)
GENEWIZ possesses extensive experience with targeted assay design, which has been demonstrated with the development of multiple in-house, proprietary panels optimized for specific applications (e.g., OncoGxOne™ Discovery cancer panels, which are optimized for cancer research).
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Some areas where GENEWIZ excels include but are not limited to:

  • Proven experience with the complexities of assay design
  • Expertise for project consultations and technical support
  • Experience and availability of all major targeting technologies
  • Proprietary, advanced bioinformatics pipelines to discover copy number variations (CNVs)
  • Proprietary, advanced bioinformatics pipelines to discover gene rearrangements
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Some possible applications include but are not limited to:

  • Discovery of common and rare genetic mutations, including point mutations, insertions/deletions (Indels), copy number variations (CNVs), and gene rearrangements.
  • Characterization of genetic associations to a particular phenotype, such as a disease state or a drug response.
  • Typing and classification of cell lines, including both eukaryotic and prokaryotic cells.
  • Antibody discovery, including from both phage display libraries and in vitro selection.
  • Genetic testing, such as pharmacogenomics or oncology assays.

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Please contact a project manager at NGS@genewiz.com for more information.

CANCER PANELS

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Cancer panels are pre-designed gene panels that use next generation sequencing to selectively sequence genes specific to cancer.
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Applications can include but are not limited to:

  • Analyzing cancer mechanisms
  • Discovering rare somatic mutations, including cancer drivers
  • Biomarker discovery
  • Therapeutic target discovery
  • Patient stratification in clinical trials
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    Whole genome sequencing requires an extremely high amount of sequencing throughput to generate even a moderate depth of coverage. The data generated, while comprehensive, will not allow the detection of mutations with as much sensitivity as a targeted approach.

    Exome sequencing is a targeted approach that targets approximately 1-2% of the whole genome. As a result, you can generate more data, and therefore a higher depth of coverage to achieve more sensitivity.

    Cancer panels target only a small percentage of what exome sequencing targets. The depth of coverage is therefore compounded even more, which makes the sensitivity of the assay high enough to effectively detect even the very rare mutations. This is crucial for cancer, because somatic mutations--which have been demonstrated to often be cancer drivers--tend to occur at a very low frequency.

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    There are primarily two types of cancer panels that GENEWIZ offers:

    1. Hot Spot Cancer Panels

    2. OncoGxOne™ Discovery Cancer Panels

    The TruSeq Amplicon Cancer Panel and the Ion AmpliSeq™ Cancer Hotspot Panel are Hot Spot Cancer Panels.

    Hot Spot Cancer Panels target regions of 48-50 genes that have been well-characterized as mutational hot spots. This is often a very small portion of the exons of those genes. The genes assayed are general to a number of different cancer types. The technology utilized for targeting is amplicon sequencing, which hybridizes pre-designed primers to flank the regions of interest directly to unfragmented genomic DNA in a highly multiplexed PCR reaction. Hot Spot Cancer Panels can provide information about point mutations and small insertions/deletions (indels).

    OncoGxOne™ Discovery cancer panels target all exons, all UTRs, and introns for which there are known translocation breakpoints of ~150-400 genes. The precise number of genes varies between the 19 panels. Each panel is specific to one cancer type. The technology utilized for targeting is targeted enrichment, which hybridizes pre-designed biotinylated baits to a fully prepared genomic library in order to pull down the regions of interest. OncoGxOne™ Discovery cancer panels can provide information about point mutations, indels, copy number variations (CNVs), and gene fusions.

    The two options have their benefits and drawbacks. For example, OncoGxOne™ Discovery panels are more comprehensive, but will take longer to generate data and will have a higher cost.

    An expert Project Manager is happy to discuss these options with you in order to determine which method would be optimal for your project and situation.


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    The major features include:

    • Specifically designed panels for each cancer type
    • Expert-curated cancer genes and genomic regions chosen by extensive literature and database mining and review
    • Most comprehensive cancer panels on the market
    • Ability to detect rare, low-frequency somatic mutations
    • Ability to detect CNVs and gene fusions in addition to point mutations and indels

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      For gene lists of the OncoGxOne™ Discovery cancer panels, and Hotspot cancer panels please contact a Project Manager at NGS@genewiz.com, or your local sales rep.

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      For all projects, raw data as FASTQ files will be delivered.

      For Hot Spot Cancer Panel projects, you will also receive a report that details point mutations and indels that were detected in your samples. This is included for all projects.

      For OncoGxOne™ Discovery cancer panels, you can optionally receive a report that details point mutations, indels, CNVs, and gene fusions.

      Additionally, you can request a custom report. For this, please specify your request in the Project Description section of the form.

      If you have any additional questions, please contact us at NGS@genewiz.com, and an expert Project Manager will be happy to work with you.

      OncoGxOne™ Discovery Cancer Panels

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      OncoGxOne Discovery cancer panels are GENEWIZ proprietary gene panels, specifically designed for comprehensively assaying genomic aberrations for each major type of cancer.
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      OncoGxOne Discovery cancer panels have many potential applications, including, but not limited to:
      • Cancer mechanism studies; e.g., identifying novel driving mutations
      • Therapeutic target discovery
      • Biomarker discovery
      • Patient stratification in clinical trials
      • Information for personalized cancer treatment
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        The major features of OncoGxOne Discovery cancer panels include:
        • Specifically designed panels for each cancer type
        • Expert-curated cancer genes and genomic regions chosen by extensive literature and database mining and review
        • More comprehensive than any other commercially available cancer-specific panels
        • Ability to detect low frequency variations
        • Ability to detect all four types of genomic aberrations (point mutations, Indels, gene fusions, CNVs)
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          A typical OncoGxOne Discovery cancer panel contains approximately 250 cancer-specific genes (including all known cancer driver genes), varying from panel to panel, ranging from ~150 to ~350 genes. The cancer gene selection for each panel is done via very extensive literature and database mining, followed by expert-curation and extensive review to ensure the scientific validity, comprehensiveness, and relevance. The data sources used for data mining include all major cancer gene-containing databases, such as COSMIC, CGC, TCGA, CGAP/Mitelman, OMIM, HMD, TTD, and DrugBank, etc., publicly available cancer panels, and major publications.
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          Currently, GENEWIZ is ready to assay the following 19 major cancer types:
          • Bladder Cancer
          • Brain Cancer
          • Breast Cancer
          • Cervical Cancer
          • Colorectal Cancer
          • Endometrial Cancer
          • Esophageal Cancer
          • Gastric Cancer
          • Head & Neck Cancer
          • Kidney Cancer
          • Leukemia
          • Liver Cancer
          • Lung Cancer
          • Lymphoma
          • Melanoma
          • Ovarian Cancer
          • Pancreatic Cancer
          • Prostate Cancer
          • Thyroid Cancer
          Upon request, GENEWIZ can also develop assays for other cancer types.
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          • Faster turnaround time
          • Lower cost
          • Detects lower-frequency variations
          • Detects gene fusions occurring in intron regions
          • Detects CNV more accurately
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          Specifically designed: Each OncoGxOne™ Discovery cancer panel is specifically designed to assay a distinct cancer, while the off-the-shelf cancer panels are general gene panels, containing many genes which may not be relevant to the cancer type of interest.

          More comprehensive: A typical OncoGxOne™ Discovery cancer panel contains ~250 cancer genes, while the off-the-shelf cancer panels contain only ~50 genes. In addition, among these ~50 genes, many may not be relevant to the cancer type of interest. 

          GENEWIZ OncoGxOne™ Discovery cancer panels assay gene exons and can be customized to assay specific intron regions by request. In contrast, the off-the-shelf cancer panels only assay limited numbers of hot spots. 

          Detects more types of genomic aberrations: Because of our proprietary panel design and bioinformatics techniques, OncoGxOne™ Discovery cancer panels can detect all four types of genomics alterations (point mutation, Indel, gene fusion, CNV), while the off-the-shelf cancer panels can only detect point mutations and Indels.

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          GENEWIZ uses the SureSelect probe-based capture assay for targeted sequence enrichment. We use MiSeq/HiSeq for data generation.

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          In order to detect gene fusions, GENEWIZ designs SureSelect probes near every potential known fusion site, and uses effective bioinformatics techniques to identify the gene fusion locations and partners. To assess the CNV (copy number variations) detection, we use control genes and "background controls" generated from the pooled customer sample data, together with proprietary bioinformatics methods. We can also use control samples (usually a blood sample, provided by customer) from the same subject to help improve the accuracy. In addition, we provide qPCR service as another CNV assay method for further verification.
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          Yes, we can provide the gene lists for all of our cancer panels to potential customers. We can also highlight those genes with their intron regions covered. The gene lists will be regularly updated, and we can include any additional genes upon customers' request.

          Exome Sequencing

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          The exome is defined here as all coding regions (exons) in the whole human genome.
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          Exome sequencing is the targeted enrichment and subsequent sequencing of the whole exome. Exome sequencing is potentially the most powerful tool available to the research community for the identification of genetic variations associated with a phenotype, such as a disease.
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          Whole genome sequencing requires an extremely high amount of sequencing throughput to generate even a moderate depth of coverage. The data generated, while comprehensive, will not allow detection of mutations with as much sensitivity as a targeted approach. Paired with current sequencing technologies, exome sequencing is the most cost-effective and efficient solution.

          A large portion of relevant mutations occur in the exome. In fact, the exome contains as many as 85% of disease-related mutations. Covering less than 2% of the whole genome, exome sequencing requires only 1/50th of the sequencing throughput to generate the same depth of coverage. The lower sequencing throughput required by exome sequencing provides flexible experimental options:

          1. Maintain the same depth of coverage and multiplex more samples into the same lane, significantly decreasing total project cost;

          2. Increase the depth of coverage to facilitate the detection of rare, low-frequency mutations;

          3. Any combination of 1 and 2.

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          GENEWIZ has proven our steadfast commitment to superior service throughout the years, becoming established as a global leader in DNA sequencing and genomics. Further, we have extensive experience and expertise in next generation sequencing techniques, including exome sequencing. An expert Project Manager can assist you in determining specifications for optimizing your project.

          16S MetaVx™ Sequencing

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          16S metagenomics is a technique that uses next generation amplicon sequencing to target specific hypervariable regions of the 16S rRNA gene.
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          16S metagenomics is used to identify the type and relative abundance of bacterial and archaeal species within a sample. This is a very effective technique when dealing with heterogeneous samples, such as soil or gut/other microbiome.
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          Current 16S metagenomics techniques use a single primer pair to target the hypervariable V4 region of the 16S rRNA gene. 16S MetaVx™ improves on this with a unique primer design that targets the hypervariable V3, V4, and V5 regions of the 16S rRNA gene.

          In an analysis comparing 16S MetaVx™ to current 16S metagenomics, 16S MetaVx™ demonstrated a higher sensitivity and specificity, identifying more bacterial and archaeal genera consistently across numerous samples.

          Additionally, current 16S metagenomics techniques require 15% or more DNA spike-in in order to combat the low sequence diversity inherent in their amplicon designs. This sequesters a significant amount of sequencing throughput with off-target coverage. Due the unique design of 16S MetaVx™, no DNA spike-in is required, dedicating the full sequencing throughput to the target regions only.

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          Please contact project management to see the results of a side-by-side analysis, where identical samples were sequenced under identical conditions using the two approaches. In every case, 16S MetaVx™ showed superior results.
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          A pie chart detailing the type and relative abundance of bacterial and archaeal species will be delivered along with the raw data.

          Have a specific question?

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