The Genomics Technology Core has provided Illumina next generation sequencing services to the UM system 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 currently operates a NovaSeq and MiSeq.
Library preparation services are offered by the Genomics 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 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
- Methyl-Seq
Non-standard library construction requests will be considered and accepted on an individual basis.
Core staff are available to assist with experimental design and to answer questions related to Illumina technology. Arrangements for a meeting to discuss new or existing projects can be made by sending an email to Nathan Bivens, core director.
Assistance can also be provided by the director for grant proposal budgets and a letter of support.
New to next-generation sequencing? Learn the basics with Illumina's beginners's guide to NGS.
The MUGTC has developed calculators to assist with the planning of sequence requirements for different applications.
Prior to sample submission and services performed, a project must be initiated with the core director and involves the following steps.
- Contact the core director to discuss project details. Several parameters are needed to quote a project and establish the scope of the work to be performed.
- Library prep type (RNA-Seq (poly A enrichment or rRNA depletion), small RNA, DNA PCR-free, WGBS)
- Organism and tissue source
- Number of DNA/RNA samples being submitted
- Number of sequence reads or Gb of coverage per sample to be generated.
- MO code for project charges.
- The core director will prepare a Project Authorization Form (PAF) outlining the services and costs. The form must be signed and returned before services are started.
- A Project Information Form should be completed and submitted by email prior to sample submission.
General Considerations:
- Submit samples only after paperwork has been completed.
- Clearly label tubes. Names must match those given on the project information form.
- Low binding microcentrifuge tubes (0.5 or 1.5 mL) are preferred for storing high quality nucleic acids.
Application Specific Requirements:
Library Type | Library Prep Input Range |
Submission Concentration |
Recommended Buffer |
Special Instructions |
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Genomic | 50 - 500 ng | 25- 200 ng/µl | EB buffer; 10 mM Tris-Cl (pH 8.5) | An image of 100 ng of genomic DNA following agarose gel electrophoresis and ethidium bromide staining will be provided by the researcher to demonstrate gDNA integrity. | |
Methyl-Seq | 10 - 200 ng | 20- 100 ng/µl | EB buffer; 10 mM Tris-Cl (pH 8.5) | An image of 100 ng of genomic DNA following agarose gel electrophoresis and ethidium bromide staining will be provided by the researcher to demonstrate gDNA integrity. | |
Chip-Seq | 1 - 10 ng | <50 µl total | EB buffer; 10 mM Tris-Cl (pH 8.5) | ||
Total RNA Stranded (rRNA depletion) | 100 - 1000 ng | 50 -200 ng/µl volume ≥15 µl | nuclease free water |
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mRNA Stranded (poly A enrichment) | 25 - 1000 ng | 50 - 200 ng/µl volume ≥15 µl | nuclease free water |
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SMARTer Ultra Low RNA(Takara) | 1 - 10 ng | <10 µl total | nuclease free water | Discuss with Genomics Technology Core Facility staff if working with less than 1 ng. | |
small RNA | 100-1000 ng | 50 - 200 ng/µl total RNA | nuclease free water |
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Library Type | Sequence Depth/Coverage* | Sequence Type | Read Length | ||
RNA-Seq (poly A enrichment or rRNA depletion) | 50 million read pairs per sample | Paired-end, UDI** | 50 or 100 bases | ||
Whole Exome Library | 100x coverage | Paired-end, UDI** | 100 bases | ||
DNA PCR-free Library | 15x-30x coverage, *depends on application | Paired-end, UDI** | 150 bases | ||
Methyl-Seq | 15x-30x coverage | Paired-end, UDI** | 150 bases | ||
Small RNA Library | 10-20 million reads per sample | Single end, single index | 75 bases | ||
* minimum read count recommended by MUGTC; increased sequencing depth may be desired for some studies
** Unique,dual-indexing is used to construct libraries types with non-repeating indexes. The MUGTC 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 MUGTC employs Illumina's unique 96 dual-indexing system to mitigate the issue.
NovaSeq 6000 | Data Output (Gb) | Single-end Reads |   |
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MiSeq | Data Output (Gb) | Single-end Reads |   |
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The shared NovaSeq service is a novel approach to provide deeply discounted sequencing costs for RNA-Seq and whole genome projects by leveraging the scaled up throughput of the NovaSeq 6000. The service lowers costs by batching samples for library preparation and sequencing on a single NovaSeq flowcell. Since batching of samples is required, there may be a longer turn around time than the typical 4 weeks. The savings, however, will be significant for projects with low sample counts. For example, a project of 12 sample for RNA-Seq would cost $511 per sample if processed as an individual group of samples on a NovaSeq SP flow cell. The $292 cost of the shared service is a 43% savings.
 
Library Type | Sequence Depth/Coverage | Sequence Type** | Cost | ||
RNA-Seq (poly A enrichment) | 50 million read pairs per sample | Paired-end, 100 bases | $234 /sample | ||
RNA-Seq (rRNA depletion) | 50 million read pairs per sample | Paired-end, 100 bases | $309 /sample | ||
Whole Genome | 90 Gb per sample | Paired-end, 150 | $745 /sample |
** Unique,dual-indexing is used to construct libraries types with non-repeating indexes. The MUGTC 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 MUGTC employs Illumina's unique 96 dual indexing system to mitigate the issue.
DNA Library | Fee |
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RNA Library | Fee |
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Epigenetic Library | Fee |
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Targeted Amplicon Library | Fee |
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NovaSeq 6000 | Fee |
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MiSeq | Fee |
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New to next-generation sequencing? Learn the basics with Illumina's beginners's guide to NGS.
Manuals
- Illumina Stranded mRNA Prep Ligation Reference Guide - Protocol for conversion of mRNA in a total RNA sample into an Illumina library of known strand origin using the reagents provided in an Illumina Stranded mRNA Prep Ligation workflow.
- Illumina DNA Prep Reference Guide - Protocol for the preparation of up to 96 DNA libraries starting from genomic DNA (gDNA) using the Illumina DNA Prep Tagmentation workflow.
- Illumina DNA Prep with Enrichment - The fastest and most flexible targeted sequencing solution for DNA in the Illumina library prep portfolio.
- NEBNext Small RNA Library Prep Set for Illumina Protocol - Protocol for preparation of small RNA libraries from total RNA or purified small RNA using a NEBNext Small RNA Library Prep workflow.
- NEBNext Ultra II DNA Library Prep Protocol - Protocol for use with NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, E7103)
- NEBNext Enzymatic Methyl-seq Kit Manual - The Enzymatic Methyl-seq kit (EM-seq) for Illumina contains all the components needed to make libraries that are enzymatically modified to detect 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC).
Technotes
- Illumina Adapter Sequences Document - Oligonucleotide (oligo) sequences of Illumina adapters used in library prep kits.
- Estimating Sequencing Coverage - Before starting a sequencing experiment, you should know the depth of sequencing you want to achieve. This Technical Note helps you estimate that coverage.
- Adapter Trimming Sequences - The recommended sequences to use for each Illumina kit.
- NEB Small RNA Adapter trimming sequences - The recommended sequences to use for trimming NEBNext small RNA data.
- Understanding unique dual indexes (UDI) - Unique dual indexing is a sequencing strategy that has distinct, unrelated index sequences for each of the i5 and i7 index reads.