Q: What is RNA-Seq?
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.
Q: How is RNA-Seq different from microarray technology?
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.
Q: How many reads do I need for my experiment, and what read length do I need?
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, fungi), 20 million reads for intermediate genomes (i.e. Drosophila, C. Elegans), 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. GENEWIZ strongly recommends following the Sample Submission Guidelines when determining the number of reads required for RNA-Seq projects. 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 read length of 50 base pairs. In most cases, 50 base pairs are sufficient to uniquely map a read to a genome. The cost of generating shorter reads is typically lower. Longer reads and paired reads help identify alternatively spliced transcripts. This level of service is available upon request.
Q: What starting material(s) does GENEWIZ accept?
At GENEWIZ, we recommend starting with total RNA extracted with a commercially available kit.
If you would like to submit cDNA or mRNA, please see the GENEWIZ RNA-Seq Sample Submission Guidelines.
Q: Can GENEWIZ assist with RNA-Seq experimental design?
GENEWIZ Project Management, a team of dedicated technical experts, is available to assist with experimental design for RNA-Seq projects. Project Management is accessible via email or by phone at 877-436-3949 from 8:00 a.m. – 6:00 p.m. (ET). In addition, GENEWIZ bioinformatics experts are available to provide assistance with your experimental design and are available to address any of technical questions. If you need help with project design or if you have any technical questions, please contact us at firstname.lastname@example.org.
Q: Does GENEWIZ have data analysis capabilities?
GENEWIZ RNA-Seq includes data analysis. GENEWIZ fully analyzes your sequencing data based on the experimental design. Gene expression or time course data analysis results include tables, graphs, and methodology details. In addition, raw data files and all intermediate results will be provided.
Q: How will GENEWIZ analyze my data?
GENEWIZ completes data analysis in accordance with your experimental design. GENEWIZ performs quality checks (QC) on the sequencing reads; maps the reads to a reference genome using a splice junction mapping tool; quantifies expression levels for genes and transcripts; performs transformation and normalization; and analyzes the results using peer-reviewed expression analysis or time course analysis algorithms.
Q: How do I submit RNA-Seq samples to GENEWIZ?
Please complete the GENEWIZ RNA-Seq Sample Submission Form, and send your samples on dry ice to:
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.
Q: Does GENEWIZ accept cDNA as a starting material? If so, how much cDNA
Yes, GENEWIZ accepts cDNA as a starting material for RNA-Seq projects. Please note, GENEWIZ is unable to provide full quality control when using cDNA due to the small quantity of starting material. GENEWIZ requires at least 500 ng of cDNA with a concentration of ≥10 ng/µl for RNA-Seq. Please elute samples in EB buffer.
Q: Does GENEWIZ accept mRNA as a starting material for RNA-Seq? If so,
how much mRNA is required?
Yes, GENEWIZ accepts mRNA as a starting material. However, due to the small quantities of starting material, we will be unable to do a quality check before beginning the library construction process. We need at least 500 ng at a concentration ≥80 ng/µl of cDNA to start. Samples should be eluted or re-suspended in nuclease-free water.
Q: How much RNA is required for RNA-Seq projects?
The following specifications apply to total RNA samples:
Plant, bacteria, and fungus derived total RNA:
- Total amount: ≥ 20 µg
- Concentration: ≥ 500 ng/µl
- OD260/280 Range: 1.8-2.2
- Re-suspended in nuclease-free water
All other total RNA:
- Total amount: ≥ 5 µg
- Concentration: ≥ 80 ng/µl
- OD260/280 Range: 1.8-2.2
- Re-suspended in nuclease-free water
Q: Does GENEWIZ accept ready-to-load libraries? If so, what are the sample
Yes, GENEWIZ can sequence ready-to-load libraries. Please send the libraries to GENEWIZ on ice at a concentration of 10nM; please include instructions for loading with your samples. Please contact GENEWIZ RNA-Seq for additional information regarding quality metrics for RNA-Seq libraries.
Q: How does GENEWIZ prepare total RNA for next generation sequencing? Do
you use poly-A selection or rRNA depletion?
GENEWIZ generally uses a poly-A selection method to enrich RNA transcripts. 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 rRNA 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.
Q: How much sequencing data does GENEWIZ guarantee?
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 2000.
Q: What if I have less starting material than indicated in the GENEWIZ
RNA-Seq Sample Submission Guidelines?
GENEWIZ may be able to work with less starting material. Please note, sequencing results are not guaranteed in these cases.
Q: How can I monitor the progress of my RNA-Seq project?
GENEWIZ provides updates throughout the duration of your project. You will be notified of our progress every time we have significant results, including:
- Completion of QC
- Data generation
- Data loaded into bioinformatics system
- Data analysis
- Results available
Q: How can I access
my data and data analysis results?
All raw data and data analysis results are accessible online via online bioinformatics solutions. Researchers will be able to explore results; perform data mining; download data files; download final results; and download intermediate results. Upon request, GENEWIZ can also ship results.
Q: What is the difference between gene-based analysis and transcript-based
Gene-based analysis quantifies expression based on the entire gene (all splice forms). Alternatively, transcript-based analysis quantifies each individual splice form.
Q: What is FPKM?
FPKM stands for Fragments Per Kilobase of exon per Million fragments mapped. FPKM is used to normalize differences in intensities due to the length of individual transcripts. Original descriptions and quantification information are available here.
Q: What is raw hit count?
Raw hit count is acquired by counting the number of reads non-ambiguously aligned to a particular genomic feature, typically a gene or transcript.
Q: Why do you recommend using raw hit count?
When experiments compare expression between samples, a comparison is made between features of the same length, so normalization by feature length is not necessary. Furthermore, algorithms utilizing FPKM quantification are transcript-based; when these algorithms are unable to un-ambiguously identify a transcript, the expression value of the gene is set to 0, which causes loss of gene-based information.
Q: What types of data analysis can GENEWIZ provide?
In addition to RNA-Seq, GENEWIZ offers fully integrated pipelines for ChIP-Seq; miRNA-Seq; SNP detection; Exome; and full genome.
Q: Will my RNA-Seq data be secure at GENEWIZ?
Client confidentiality and protection of Intellectual Property (IP) is 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.