Illumina NextSeq Series Next-Generation Sequencing
NextSeq 500 systems delivers the power of high-throughput sequencing with the simplicity of a desktop sequencer. Its fast, integrated, sample-to-results workflow enables rapid sequencing of whole genome, exomes, targeted panels, and transcriptomes in a single run, with the flexibility to switch to lower-throughput sequencing as needed. The systems fit seamlessly into research laboratories, with no need for specialized equipment. Illumina scientists are available at every point along the way with support and guidance, enabling researchers to focus on making the next breakthrough discovery
What are the key features of NextSeq 500 for genomic applications?
The NextSeq 500 offers the right sequencer for any project size and sequencing throughput, providing users with optimal operational efficiency. It is the only desktop sequencing system capable of sequencing a high-coverage (30×) whole human genome in one run. The NextSeq 500 also delivers a one-day turnaround for numerous popular sequencing applications. With these instruments, researchers can sequence a broad range of samples per run:
- 1–16 exomes
- 1–20 transcriptomes
- 6–96 targeted panels
- 12–40 gene expression profiling samples
The NextSeq 500 is easily configured providing researchers with scalability to handle low to high-throughput project sizes for maximum operational efficiency. Based on sample volume, complexity and coverage needs, researchers can choose between 2 flow cell configurations (High Output and Mid Output), easily shifting from low- to higher-throughput.
What is the advantage of the NextSeq 500™ System compared to microarray for genomic applications?
The data collected by microarray are based on the fluorescence signal intensities (analogue data) from known sequenced oligo probes. Due to technical limitations, microarray data have issues regarding specificity, discrimination and sensitivity. However, the NextSeq series™ NGS generates genome-wide sequence data as “digital data” in the resolution of single nucleotide and has no specificity, discrimination and sensitivity issues. The sequence data easily can be used to identify single nucleotide deletion, insertion, polymorphism and translocation and copy a number of changes on the DNA level. The sequence data generated from RNA in transcriptome-wide analysis could easily be used to identify transcript copy number variation between samples, splicing variants, single nucleotide change by RNA editing and discovery for all transcribed non-coding RNA transcriptome-wide.
What do I need to do for my research project in order to use the NextSeq 500™ System NGS technology?
Please design and plan your experiments well to address your questions scientifically. The next step is to collect high-quality DNA or RNA material as planned. You may place an online iLab request (https://mdanderson.ilabsolutions.com/sc/3659/mdacc-sequencing-and-ncrna-program/) to the ncRNA Program and submit your samples for sequence data production.
When should I expect to receive the sequence raw data and analyzed data?
The NextSeq 500 supports several data analysis options. Integrated instrument computers perform base calling and quality scoring as raw data. Sequencing run data can be run through a wide range of open-source or commercial pipelines developed for Illumina data .fastq file, or instantly transferred, analyzed, and stored securely in BaseSpace® (Cloud or Onsite), the Illumina genomics computing environment. BaseSpace downstream data analysis includes alignment and variant detection, annotation, visualization, and interpretation. BaseSpace also includes Illumina data analysis apps for exome, transcriptome, whole-genome, and somatic variant calling. Thanks to industry-standard data formats, third-party developers have created a rich ecosystem of commercial and open-source tools for more extensive downstream data analysis.
Data analysis will be fully supported by moonshot genomic bioinformatics team led by Dr. Jianhua Zhang (713-792-6807) in the form of paid service. Interested parties are encouraged to communicate with the team regarding the study and analysis expected to be done.
Life Technologies-Ion Proton Sequencing
The Ion Proton™ System is the first benchtop non-imaging semiconductor sequencing system capable of human-targeted genome, exome, or transcriptome sequencing in a few hours—with DNA-to-variants called in a single day. Ion Ampliseq chemistry enable to work on FFPE samples to sequence targeted cancer-related gene panels for DNA mutation and Copy Number Variation (CNV) detection and gene mRNA expression profiling.
The system combines semiconductor sequencing technology with natural biochemistry to directly translate chemical information into digital data. By leveraging the exponential improvements in the semiconductor industry (known as Moore’s Law), the Ion Proton™ Sequencing System provides an unprecedented level of scalability and flexibility to support a broad range of high throughput sequencing applications, ranging from human-scale genome to exome to transcriptome sequencing. The system’s use of the simplest natural sequencing chemistry eliminates the need for expensive optics and complex sequencing chemistries, resulting in a highly affordable sequencing system to own and operate. Real-time, direct, electrical detection of sequencing, combined with the enormous amount of computing power in both the Ion Proton™ Sequencer and Ion Proton™ Torrent Server, enables generation of high quality sequencing results from DNA library to variants in a single day.
Features of the Ion Proton™ System:
- Fastest high-throughput next-generation sequencing workflow with the fastest sequencing run-times of 2–4 hours on the Ion PI™ Chip
- Highest number of complete sequencing runs per week, with a simple and automated workflow (when used with the Ion OneTouch™ 2 System)
- Simple semiconductor sequencing workflows with robust and simple hardware that can be relied upon—no cameras, no optics, and no lasers
- Scalable, high throughput sequencing capabilities enabling up to 200-base single reads and flexible library options (e.g., fragment gDNA, targeted/exome, RNA)
- Small benchtop footprint and mounting capability (with optional rack) (two Ion Proton™ systems per rack), to deliver the only benchtop genome center
- Low-cost platform and attractively priced semiconductor chips and reagents for a range of applications
- Proven Ion semiconductor sequencing requires no complex optics and employs natural nucleotides to deliver highly accurate variant detection, uniformity of coverage, and sensitivity to detect low frequency variants
- Range of fast and simple library solutions and kits with low-input requirements for a variety of applications, such as genomic sequencing, exome sequencing, sequencing sets of genes, and RNA sequencing
- Complete end-to-end solution from base calls to variants with the Proton™ Torrent Server and Torrent Suite Software v3.0
- Simple and integrated tools for tertiary data analysis with Ion Reporter™ Software for DNA variation analysis across single, paired, or trio samples
What is the difference in total RNA required for mRNA and microRNA expression profiling?
General speaking, microarray requires high-quality intact total RNA as starting material for target preparation in mRNA profiling. For mRNA expression profiling, we recommend RNEasy Column purification, after RNA isolation by TRIzol®, to remove small RNA and contaminated protein. For microRNA expression profiling, however, we need whole total RNA without any column purification because mature microRNA in sizes 19~22nt and their precursors in sizes 60~110nt can be lost after column purification.
What types of oligo microarrays are available from the ncRNA Program?
The microarrays utilized in the ncRNA Program are oligo-based Affymetrix and in-house custom arrays. The ncRNA Program provides expression profiling and SNP genotyping on all Affymetrix products commercially available. In addition, we build in-house oligo microarrays for ncRNA expression profiling, such as microRNA and tsRNA, ultraconserved and Pyknon ncRNA expression profiling.
What do I need to provide?
The research investigator provides high-quality total RNA only. The ncRNA Program takes care of the rest of target preparation, hybridization, chip post-hybridization signal detection, chip scanning, chip girding, data crunching, preliminary data analysis and data interpretation, if needed.
What expression profiling technology is offered by the ncRNA Program?
The technology for expression profiling is a single-color system on both Affymetrix and custom arrays. The oligo probes are in situ synthesis (Affymetrix 25mer) or spotted (custom ncRNA array 40mer) and immobilized on the chip. The biological testing sample is processed further by linear amplification and biotin-labeling during in vitro transcription (IVT) to generate a single strand antisense RNA (aRNA) as a target for chip hybridization. The labeled aRNA is on a single chip for hybridization and signal detection by Streptavidin-phycocerithrine in the post-hybridization process. The median normalized data from control and testing samples are compared to each other in fold change for further identification of differentially expressed genes.
How do I design my microarray experiments?
Due to the natural variation of samples collected from batches and individuals technically and biologically, it is most economical to prepare multiple biological samples and not request multiple chips for the same mRNA sample. Keep in mind that with 20K~45K spots, some spots may not hybridize equally on your control and treated chips because of technical reasons (minor defects, fiber, etc.). For this reason, it is essential that you repeat your experiment initially in duplicates. You can then focus your initial analysis on the mRNAs that are altered in both experiments. If you find that this initial experiment provides data that is promising, you most likely want to perform a third experiment to facilitate statistical studies.
It is important to address that it is not efficient to perform only one control and treated chip without a duplication because you are likely to spend significant time analyzing data that are artificial. Typically, the ncRNA Program receives samples comparing testing samples from control sample. Of the thousands of samples processed, we have noticed two strategies that most labs take:
- The first strategy involves repeating experiments only once and then use hits common on both experiments for RT-PCR and whole mount studies and really have no plan to initially publish their chip results. These groups validated most of their hits and have papers in progress. Keep in mind that this approach certainly will miss hits because the number of duplicate experiments is too low.
- The second strategy involves doing three to four replicates followed by statistical analysis performed either at the investigator’s own institution or through our collaboration with the bioinformatics group. This approach allows you to identify a comprehensive list of statistically significant hits prior to a more detailed biological analysis.
While we recognize that many of you have limited resources that motivate opting for the first strategy, we strongly recommend the second strategy despite its higher initial cost.
If you have questions about your experimental design of for more information about bioinformatics analysis, please contact Chang-gong Liu, Ph.D., prior to performing your experiment.
How do I isolate RNA for use in mRNA and microRNA microarray experiments?
The main objective is to generate RNA that is of high quality and sufficiently concentrated for use in a microarray experiment. Many researchers find that RNA isolation with TRIzol reagent gives good quality and quantity of RNA for these experiments. However, each type of starting material may generate RNA of different quality and no one technique is likely to work for all samples. Your data quality depends on the total RNA quality provided by you.
How do I evaluate the RNA quality for expression profiling on microarray?
RNA quality can be measured by Agilent Bioanalyzer 2100 with RNA Index Number (RIN), and quantitated by Nanodrop 1000 as well as by calculating the A260/A280 ratio.
The spectrophotometric ratio also will give an indication of the purity and integrity of the RNA and should be as close to 2.0 as possible. Generally, ratios less than 1.7 indicate that the RNA may be contaminated with other material and should be re-purified, perhaps run through a column.
What should be the concentration of the total RNA sample for submission to the ncRNA Program?
The concentration of the testing RNA should be in range 100-500ng/µl minimal in RNase-free H2O. The ncRNA Program requires ~2-3 µg of total RNA for each testing sample.
What steps should I take prior to submission of samples to the ncRNA Program?
Please ensure that:
- The RNA quality has been properly evaluated and provide an agarose gel image with the RNA samples or you may request that the ncRNA Program provide a quality check on the Agilent Bioanalyzer 2100
- Please do online iLab service request: https://mdanderson.ilabsolutions.com/sc/3659/mdacc-sequencing-and-ncrna-program/
How should I address and send the RNA?
External users should send the RNA on dry ice via an overnight carrier to the address listed on our contacts page. All RNAs will be stored in -80°C freezer at the facility until processed.
What are the costs associated with a microarray experiment?
We are a fee-for-service program and offer the same prices to the communities of MD Anderson network includes UT system Institutions Baylor College of Medicine, Methodist, University of Houston and Gulf Coast Consortium. External institutions out of network will charge 60% as overhead. If you have more questions, please contact Chang-gong Liu, Ph.D., for detailed pricing.
After completion of an experiment, what information and data can I expect to receive?
You will get back all raw data file as *cel files, *data files, *tif files and *gpr files. Also, the raw data that have been crunched and exported from the image data and preliminary analyzed data with fold change of listed differentially expressed genes will be provided in an Excel spreadsheet.
How do I retrieve the data?
Data can be retrieved in several ways. Most commonly, an ftp site (username and password protected) is established on the computer of the cancer center server with a private account for your data. Your data will be sent to the IP address of the relevant computer and you may access the site online to download the data to your own computer.
What is the turnaround time for experiments?
Work in the ncRNA Program is completed on a first come, first served basis. Generally, microarray experiments can be completed within one week of receipt of a small number of samples. However, the turnaround time for larger projects will be based on the number of samples and the queue in the laboratory.