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.
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.
Attn: NGS Laboratory
115 Corporate Boulevard
South Plainfield, NJ 07080
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.
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.
The following specifications apply to total RNA samples:
How does GENEWIZ prepare total RNA for next generation sequencing? Do you use poly-A selection or rRNA depletion?
For prokaryotic samples, GENEWIZ depletes the rRNA for all projects.
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.
What if I have less starting material than indicated in the GENEWIZ RNA-Seq Sample Submission Guidelines?
GENEWIZ provides updates upon sample receipt and upon confirmation of quality control results. Subsequently, GENEWIZ provides weekly updates throughout the duration of your project.
Raw hit count is acquired by counting the number of reads non-ambiguously aligned to a particular genomic feature, typically a gene or transcript.
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.
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 is an application of next generation sequencing where you enrich regions of the genome that you are interested in sequencing.
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.
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.
What are some advantages and disadvantages of all of these different types of targeted resequencing, and how do you choose which one to go with for a particular project?
The assay design process can range from relatively straightforward to extremely complex.
Some key elements of the design process include:
Some possible applications include but are not limited to:
Please contact a project manager at NGS@genewiz.com for more information.
Applications can include but are not limited to:
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.
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.
The major features include:
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.
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.
How many genes are included in each OncoGXOne Discovery cancer panel? How do you select the cancer gene for each panel?
What are the major advantages of OncoGxOne Discovery cancer panels vs. the off-the-shelf commercial cancer panels from Illumina and Ion Torrent?
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.
GENEWIZ uses the SureSelect probe-based capture assay for targeted sequence enrichment. We use MiSeq/HiSeq for data generation.
Detection of point mutations and Indels are relatively straightforward, but how do you detect gene fusion and CNV?
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.
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.
Has there been any analysis to show the 16S MetaVx™ design provides improved performance in comparison to current 16S metagenomics techniques?