The DNA Core Facility has provided Illumina next generation sequencing services since 2008. Core staff are experienced with RNA/DNA QC techniques, a multitude of library construction methods, and data generation on Illumina instruments.
The facility offers free consultation for researchers in the grant application process or project initiation phase. Staff can discuss current technology capabilities, prepare cost estimates, and provide letters of support. Additional literature and information on applications, as well as selected publications, can be obtained from the Illumina website.
Fees for all NGS services are listed on the DNACF Fee List
Contact the DNA Core for additional information on conducting a sequencing project with the facility.
Library Construction Services
Library preparation services are offered by the DNA Core with >2000 libraries constructed in-house annually . Staff are experienced in constructing libraries from a diverse set of biological systems using varied methods. The DNA Core currently offers the following as standard library construction services.
- mRNA Stranded Library (poly A enrichment)
- Total RNA Stranded Library (rRNA depletion)
- Small RNA Library
- Ultra Low Input RNA Library
- DNA PCR-free Library
- Whole Genome Bisulfite Library (WGBS)
- Methylated DNA Enrichment Library
- Target Amplicon Libraries (16S, ITS, etc.)
Non-standard library construction requests will be considered and accepted on an individual basis.
Researchers may submit libraries prepared in their own labs. The DNA Core recommends researchers discuss with core staff the preparation of libraries to ensure successful sequencing.
The Illumina sequencing platform has the capability to pool samples in a single lane to reduce the cost of sequencing. Sample pooling, or multiplexing, is accomplished with a barcode sequence incorporated in to each fragment during library construction that is used to sort reads post-run. The DNACF is familiar with the issue referred to as “index hopping” which results in the misassignment of reads. The issue is more pronounced in Illumina’s exclusion amplification chemistry and patterned flowcells (i.e. NovaSeq). The DNACF employs Illumina’s unique 96 dual indexing system to mitigate the issue.
Submission Guidelines for Library Construction
|Library Type||Quantity Required||Concentration||Recommended
|DNA PCR-free (TruSeq)||1000 - 2000 ng||40- 200 ng/ul||EB buffer; 10mM Tris-Cl (pH 8.5)||An image of 100ng of genomic DNA following agarose gel electrophoresis and ethidium bromide staining will be provided by the researcher to demonstrate gDNA integrity.|
|Whole Genome Bisulfite Sequencing||1000 ng||100- 200 ng/ul||EB buffer; 10mM Tris-Cl (pH 8.5)||An image of 100ng of genomic DNA following agarose gel electrophoresis and ethidium bromide staining will be provided by the researcher to demonstrate gDNA integrity.|
|Methylated DNA Enrichment||1000 ng||100- 200 ng/ul||EB buffer; 10mM Tris-Cl (pH 8.5)||An image of 100ng of genomic DNA following agarose gel electrophoresis and ethidium bromide staining will be provided by the researcher to demonstrate gDNA integrity.|
|Chip-Seq||5 - 10 ng||<50 ul total||EB buffer; 10mM Tris-Cl (pH 8.5)|
|Total RNA Stranded (rRNA depletion)||500 - 1000 ng||50 -200 ng/ul volume ≥15ul||nuclease free water||
|mRNA Stranded (poly A enrichment)||100 - 1000 ng||50 - 200 ng/ul volume ≥15ul||nuclease free water||
|SMARTer Ultra Low RNA(Takara)||1 - 10 ng||<10 ul total||nuclease free water||Discuss with DNA Core staff if working with less than 1 ng.|
|small RNA||1500 ng||200 - 400 ng/ul total RNA||nuclease free water||
- General Comments
- Advance arrangements should be made before dropping samples off at the DNACF.
- Submit samples only after paperwork has been completed.
- Clearly label tubes. Names must match those given on the library information form.
- Low binding microcentrifuge tubes (0.5 or 1.5mL) are preferred for storing high quality nucleic acids.
- Submission of Sequence Ready Libraries
- 20ul of 10nM stock library
- Indicate the type of library, fragment size and adapters used. This information will be provided on the Library Information Form.
- Fragment size profile generated by Agilent Bioanalyzer (or similar instrument).
- A Qubit quantitation assay will be performed on each library.
- NextSeq 500
- The NextSeq 500 provides high-throughput sequencing at increased speeds and lower cost. The faster run times are achieved with use of Illumina's new two-channel SBS technology which requires only two images to capture all four base calls.
Review the following Illumina technology notes to understand the difference between two-channel and four-channel SBS sequencing.     Learn More »
- Standard sequence read lengths of 75 and 150 bases.
- Single or paired-end reads
- ~800 million quality paired-end reads per flow cell
- Maximum data yield of 120Gb per flow cell
- Greater than 75% of bases >Q30 on a 2x150 run
- The MiSeq is a DNA sequencing platform capable of generating paired-end, 300 base read lengths. The single lane flow cell design, on-board clustering and five minute cycle times allow for rapid sequencing times(~48 hours for a 2x250 run).
- Standard sequence read lengths of 75, 150, 250 and 300 bases
- Single or paired-end reads
- 25 million quality reads per lane
- Maximum data yield of ~15 Gb
- Greater than 70% of bases >Q30 on a 2x300 run