The CWRU Genomics Core utilizes the highly trusted Illumina platforms for all Next-Gen Sequencing (NGS) services. The NextSeq 550 Sequencing System is a flexible high through-put benchtop sequencer with fast run times and exceptional data accuracy. It is used for all of our high- to mid- throughput projects, such as exome sequencing, whole-transcriptome, and ChIP-seq.
MiSeq desktop sequencer offers fast runs times and long read lengths. It is best for low-throughput projects, such as amplicon sequencing, 16S rRNA-seq, or small genomes. The MiSeq has the capability of running all v2 and v3 flowcells available.
Collectively, the Core is able to handle a wide range of applications and different sized projects at the same time.
| Whole Exome Sequencing | Whole Transcriptome Sequencing | Small RNA Sequencing |
| Whole Genome Sequencing | ChIP Sequencing | CRISPR Validation |
| 16S Metagenomics | Amplicon Sequencing | Targeted Sequencing |
To set up a consultation for a project, or if you have any questions, please contact the Genomics Core Director, Dr. Alexander Miron, at 216.368.2791 or at alexander.miron@case.edu. For project quotes, please contact Simone Edelheit, Core Manager, at 216.368.1887 or at sxw94@case.edu.
NGS Run Options & Outputs
View the table below to determine which platform is best suited for your project:
| NextSeq 550 High Output Flowcell | NextSeq 550 Mid Output Flowcell | MiSeq V3 FLOWCELL | |
|---|---|---|---|
| Clusters per Flowcell | 400 million | 130 million | 25 million |
| Maximum Read Length | 2x150bp | 2x150bp | 2x300bp |
| Whole Genome | 1 | n/a | n/a |
| RNA-seq per flowcell | 9-16 (25-50M per) | 2-5 (25-50M per) | n/a |
| ChIP-seq per flowcell | 9 (50M per) | 2-3 | n/a |
| Exomes per flowcell | 6-12 (50-100X coverage) | 1-3 (50-100X coverage) | n/a |
| 16s Samples with >100,000 read coverage | n/a | n/a | 192 |
| CRISPR-cas Validation | n/a | n/a | 288 |
For a list of all runs types available and pricing, please see Pricing.
Sample Submission
If you are submitting gDNA or total RNA for library prep to be completed by the Core, visit our Library Prep page for sample submission information and details.
For all submissions of already prepared libraries or final library pools, follow the instructions below for sample submission requirements. Contact the core with any questions.
Final Library Requirements:
| 1. | 2. | 3. |
|---|---|---|
|
Provide a minimum of 20ul of each Final Library or Final Library Pool and a concentration between 1.0ng/ul and 20ng/ul (by Qubit). |
Submit all samples in 1.5ml eppendorf tubes, clearly labeled on TOP of tube with sample name and user initials, and label SIDE of tube with the date. Sample names on tube and iLab request form should match. |
Large volume projects may be submitted in FULL SKIRT 96-well plates, with at least one SIDE WALL clearly labeled with project name, plate number, user name, and date. Sample names and well location will be submitted through iLab. |
Whether prepared by the user or the Core, all final libraries will undergo quality control (QC) prior to running on the Illumina sequencers. The Core will run Qubit for concentration (ng/ul) and Agilent Bioanalyzer High Sensitivity DNA chip for library quality and average fragment size (bp). If samples pass QC, they will be pooled equally and according to adapter-indexes used. The pool will be quantified using the NEBNext Library Quantification kit for Illumina. The final library pool will be diluted and denatured for sequencing following Illumina protocols. All fees for Final Library QC, pooling, and qPCR are separate and not included in the cost of a run. Additional charges to each project vary depending on which QC services are completed. For a list of all QC tests available and pricing, please see Pricing.
Run Analysis and Data Collection
Upon completion of a run, the data is automatically sent through a series of custom pipelines. FASTQ files are generated for all samples within a run and provided to the user in Illumina 1.8x format. We perform quality control (QC) of the run using software provided by Illumina as well as FastQC to confirm that high quality data is provided.
Data is released to the user once it has passed our quality checks. All NextSeq and MiSeq run data are shared with the user via secure log-in SFTP site, where files are available for download for 30 days. Data from all NextSeq and MiSeq runs are stored for six months. After six months, all NGS runs are archived to tape. We strongly recommend that all users keep a back-up of all data provided by the Core.
NGS Run Guarantees
Each Illumina NGS platform delivers a maximum output dependent upon the type of flowcell and reagents used. In practice, this output can also be affected by the DNA source and the type of library created. It is our goal to have each run collect the highest quality and quantity of data possible for the set of samples being run.
The Genomics Core will guarantee a minimum of 80% of the maximum number of possible reads as listed by Illumina based on instrument type and reagents used if the Core completes all quality control (QC) on submitted total RNA and/or DNA and performs the library prep, final library QC, pooling and qPCR. All libraries used must pass QC in order for the run to be guaranteed.
The Genomics Core will guarantee a minimum of 70% of the maximum number of possible reads as listed by Illumina based on instrument type and reagents used if the user submits final library preps that use all standard Illumina reagents (ie: primers) and the Core completes final library QC, pooling and qPCR. All libraries used must pass QC in order for the run to be guaranteed.
It is our goal to have each run collect the highest quality and quantity of data possible for the set of samples being run, however at times there are limitations. The Core cannot guarantee a minimum data output in the following cases: when a run is performed using QC data provided by the user, if any custom sequencing/indexing primers are used in replacement of Illumina provided reagents, or a run that includes experimental libraries that have not been previously tested at our core or by Illumina.