Next Generation Sequencing
- Applications
- Fees
- Instrumentation
- Sample Submission
- Data Analysis
- Policies
- Illumina Contact Information
Applications
Fees
Fees for Core Preparation of Libraries |
||
---|---|---|
APPLICATION | ON CAMPUS | OFF CAMPUS |
Stranded Total RNA Sequencing | ||
Sample QC, Library Preparation and Sequencing | $250/sample | $400/sample |
Data Storage (up to 1TB of data) | $100 | $150 |
Data Analysis | $300 | $400 |
Stranded mRNA Sequencing | ||
Sample QC, Library Preparation and Sequencing | $200/sample | $375/sample |
miRNA Sequencing | ||
Sample QC, Library Preparation and Sequencing | $150/sample | $250/sample |
ChIP/CUT&RUN Sequencing | ||
Sample QC, Library Preparation and Sequencing | $100/sample | $150/sample |
DNA Sequencing | ||
Sample QC, Library Preparation and Sequencing | $125/sample | $250/sample |
Metagenomics/Amplicon | ||
Sample QC, Library Preparation and Sequencing | $50/sample | $100/sample |
Fees for Submission of User-Prepared Libraries |
||
---|---|---|
SERVICE | ON CAMPUS | OFF CAMPUS* |
Agilent TapeStation QC and Qubit Quantification | $10/sample | $20 |
qPCR for Library Quantification | $10/sample | $20 |
Sample Pooling (up to 12 samples) | $50 | $100 |
Normalization, Denaturing and Diluting | $50 | $100 |
Sequencing Run | $150 | $300 |
Data Storage (up to 1TB of data) | $100 | $150 |
Sample Cleanup (column or bead-based) | $10/sample | $10/sample |
- A 20% discount may be applied when processing a minimum number of samples for RNA sequencing; please inquire with Core staff.
- Off campus fees apply to academic institutions only. Non-academic facilities should contact the Core for additional information.
- Regardless of the application, the investigator is responsible for purchasing the Illumina sequencing reagent kit (sequencing cartridge). The price of the sequencing reagent kit depends on the read length and desired output. Please consult with Core staff prior to purchasing.
- Samples will be put in the queue for processing ONLY after they have been received and pass all quality control measures; however, we will do our best to accommodate deadlines when possible.
Illumina Consumable (Price List as of March 2023) |
|||
---|---|---|---|
Sequencing Kit | Catalog Number | Cycle Number | ~Cost |
MiSeq Reagent Kit v2 | MS-102-2001 | 50 | $956.65 |
MiSeq Reagent Kit v2 | MS-102-2002 | 300 | $1,224.55 |
MiSeq Reagent Kit v2 | MS-102-2003 | 500 | $1,377.50 |
MiSeq Reagent Kit v3 | MS-102-3001 | 150 | $1,062.10 |
MiSeq Reagent Kit v3 | MS-102-3003 | 600 | $1,792.65 |
MiSeq Reagent Micro Kit v2 | MS-103-1002 | 300 | $514.90 |
MiSeq Reagent Nano Kit v2 | MS-103-1001 | 300 | $341.05 |
MiSeq Reagent Nano Kit v2 | MS-103-1003 | 500 | $409.45 |
NextSeq 500/550 Mid Output Kit v2.5 | 20024904 | 150 | $1,266.35 |
NextSeq 500/550 Mid Output Kit v2.5 | 20024905 | 300 | $2,039.65 |
NextSeq 500/550 High Output Kit v2.5 | 20024906 | 75 | $1,6734.70 |
NextSeq 500/550 High Output Kit v2.5 | 20024907 | 150 | $3,323.10 |
NextSeq 500/550 High Output Kit v2.5 | 20024908 | 300 | $5,325.70 |
NovaSeq 6000 SP Reagent Kit v1.5 | 20028401 | 100 | $2,207.80 |
NovaSeq 6000 SP Reagent Kit v1.5 | 20040719 | 200 | $2,891.80 |
NovaSeq 6000 SP Reagent Kit v1.5 | 20028400 | 300 | $3,154.95 |
NovaSeq 6000 SP Reagent Kit v1.5 | 20028402 | 500 | $4,416.55 |
NovaSeq 6000 S1 Reagent Kit v1.5 | 20028319 | 100 | $4,047.95 |
NovaSeq 6000 S1 Reagent Kit v1.5 | 20028318 | 200 | $5,099.60 |
NovaSeq 6000 S1 Reagent Kit v1.5 | 20028317 | 300 | $5,520.45 |
The cost above reflects a standard 5% discount provided to LSUHS users only. An additional discount of 7.5% may be provided by reaching out to Lewis Gill (lgill@illumina.com) or Cory Reier (creier@illumina.com). External users are also encouraged to reach out to Illumina to request a quote with discounted pricing.
Please consult with Core Staff or check Illumina's website for accurate pricing and a complete list of all sequencing kits available.
Sample Coverage: To determine the number of samples that can be pooled in one sequencing run, access the Illumina Sequencing Coverage Calculator. Keep in mind that the calculator uses an estimate of reads passing filter commonly found for balanced genomes (such as PhiX or the human genome). If you plan to sequence an unbalanced genome, you may have a lower number of reads passing filter, and consequently a lower output.
For more information about calculating coverage estimates, reference the technical note Estimating Sequencing Coverage or the Sequencing Coverage Calculator.
Instrumentation
Illumina MiSeq
The MiSeq is useful for focused applications such as small genome sequencing, metagenomics, amplicon sequencing and targeted gene sequencing. It is capable of generating up to 15 GB of output with 25 M sequencing reads and up to 2 x 300 bp read lengths.
Cluster generation, sequencing and analysis are all performed on the MiSeq. The sequencing process takes place on a single channel flow cell. Multiple samples can be pooled together and sequenced by using unique index barcodes during library preparation
Chemistry |
Cycles |
Read Length |
Output |
# of Single Reads |
~Run Time |
V2 |
50 |
1 x 36 bp |
≤ 610 Mb |
≤15 million |
4 hours |
50 |
2 x 25 bp |
≤ -850 Mb |
≤ 15 million |
5.5 hours |
|
300 |
2 x 150 bp |
≤ 5.1 Gb |
≤ 15 million |
24 hours |
|
500 |
2 x 250 bp |
≤ 8.5 Gb |
≤ 15 million |
39 hours |
|
V3 |
150 |
2 x 75 bp |
≤ 3.8 Gb |
≤ 25 million |
24 hours |
600 |
2 x 300 bp |
≤ 2-15 Gb |
≤ 25 million |
65 hours |
|
V2 Micro | 300 | 2 x 150 bp | ≤ 1.2 Gb | ≤ 4 million | 19 hours |
V2 Nano |
300 |
2 x 150 bp |
≤ 300 Mb |
≤ 1 million |
17 hours |
500 |
2 x 250 bp |
≤ 500 Mb |
≤ 1 million |
28 hours |
Illumina NextSeq 550
The NextSeq 550 is a more flexible system, enabling the analysis of transcriptomes, an exams. It is capable of generating up to 400 M single sequencing reads and up to 2 x 150 bp read lengths.
Mode |
Cycles |
Read Length |
Output |
# of Single Reads |
~Run Time |
---|---|---|---|---|---|
Mid Output |
150 |
2 x 75 bp |
≤ 19.5 Gb |
≤ 130 million |
15 hours |
300 |
2 x 150 bp |
≤ 39 Gb |
≤ 130 million |
26 hours |
|
High Output |
75 |
1 x 75 bp |
≤ 30 Gb |
≤ 400 million |
11 hours |
150 |
2 x 75 bp |
≤ 60 Gb |
≤ 400 million |
18 hours |
|
300 |
2 x 150 bp |
≤ 120 Gb |
≤ 400 million |
29 hours |
Illumina NovaSeq 6000
The NovaSeq 6000 is a powerful and flexible high-throughput next generation sequencer, enabling the analysis of whole genomes, single cell RNA libraries and Visium Spatial gene expression libraries. Its scalable output allows for generation of up to 6 Tb and 20 billion reads.
Mode |
Cycles |
Read Length |
Output |
# of Single Reads |
~Run Time |
---|---|---|---|---|---|
SP |
2 x 50 bp |
≤ 80 Gb |
≤ 800 million |
13 hours | |
2 x 100 bp |
≤ 167 Gb |
≤ 800 million | 19 hours | ||
2 x 150 bp |
≤ 250 Gb |
≤ 800 million | 25 hours | ||
2 x 250 bp |
≤ 400 Gb |
≤ 800 million | 38 hours | ||
S1 |
2 x 50 bp |
≤ 167 Gb |
≤ 1.6 billion |
13 hours | |
2 x 100 bp |
≤ 333 Gb |
≤ 1.6 billion |
19 hours | ||
2 x 150 bp | ≤ 500 Gb | ≤ 1.6 billion | 25 hours |
Sample Submission
Sample requirements vary depending on application. The following may be used as a guideline; however, always consult Core staff prior to sample extraction.
Application | Input | Min Conc.** | Volume for QC*** |
Total RNA-Seq | 0.3 - 1 ug | 50 ng/ul | 3 ul |
mRNA-Seq | 0.3 -1 ug | 50 ng/ul | 3 ul |
Nextera XT DNA | 1-2 ng | 0.2 ng/ul | 3 ul |
Metagenomics | 12.5-25 ng | 5 ng/ul | 3 ul |
User Prepared Libraries | ≥5 nM | 15 ul | NA |
*Please consult Core staff if your samples do not meet these minimum requirements.
**Concentration is based on validations by Core staff using a fluorometric assay.
***This volume is in addition to the volume required for processing.
Please schedule an appointment to discuss sample submission guidelines with Core staff prior to submitting samples.
- Please fill in all information on the provided sample submission form.
- Please do not submit your entire stock RNA/DNA sample to the Core. When eluting or re-suspending, make two aliquots for submission. This will prevent subjecting your sample to unnecessary freeze-thaw cycles. Make a 3 ul aliquot for QC and an additional aliquot for processing.
- Samples will be put in the processing queue only after they have been received and pass all quality control metrics.
- Use an established isolation method that produces an intact sample free of contaminants that may inhibit downstream applications. It is best to elute in nuclease-free water when possible. AVOID buffers containing EDTA and minimize carryover of salts, phenol, etc.
- Submit samples in clearly labeled 1.5 ml tubes with the submission date and sample ID. Emailing sample IDs to Core staff is also recommended.
- Prior to processing, samples will be quantitated using the appropriate Qubit fluorometric assay, and, if starting with RNA, sample integrity will be assessed on an Agilent TapeStation.
- Biological replicates of at least n=3 are always recommended to gain useful insight when performing data analysis.
Overall Sequencing Process:
- Project Consultation: During project consultation, you will meet with the Genomics Core staff to discuss your research hypothesis and experimental design. We will discuss sample requirements, replicate number, sequencing coverage, read length, and output.
- Sample Submission: You must provide Genomics Core staff with a completed sample submission form at the time of sample submission.
- Sample QC: Depending on the application, samples may be submitted to the Genomics Core as DNA, RNA, amplicons, or library. For best results, DNA and RNA should be of high quality and purity and should be intact (not degraded). We recommend that DNA and RNA samples be resuspended in nuclease-free water to avoid the presence of inhibitors. Prior to processing, we will run an Agilent TapeStation analysis to determine quality and a Qubit fluorometric assay to determine concentration.
- Library Generation: The type of libraries generated will depend on the research question being asked. The most frequently requested libraries are Illumina Stranded mRNA, Illumina Stranded Total RNA, ChIP, Nextera DNA, and amplicon.
- Library QC: Libraries are analyzed on an Agilent TapeStation to determine size and detect any contaminating products. They are then quantitated using either qPCR or a Qubit fluorometric assay (this depends on the library type).
- Illumina Sequencing: Once the libraries pass QC and concentration is determined, they are ready for sequencing on either an Illumina MiSeq, an Illumina NextSeq 550, or an Illumina NovaSeq 6000.
- Data Delivery: When the run has completed, base calls are converted to fastq files in Illumina BaseSpace Cloud. You will receive an email containing run specifications and sample information, such as number of reads per sample. You will be given access to the run and fastq files through BaseSpace.
- Bioinformatics: The Research Core Facility does not perform any data analysis. It is recommended that you reach out to the COBRE Center for Applied Immunology and Pathological Process (CAIPP) Modeling Core during the planning phase of your experiment. Reach out to Dr. Rona Scott - Rona.Scott@lsuhs.edu or Dr. Jian Wang - Jian.Wang@lsuhs.edu.
Samples will be put in the queue for processing ONLY after they have been received and pass all quality control measures.
Data Analysis
The Center of Applied Immunology and Pathological Processes (CAIPP) Modeling Core can provide further assistance with data analysis, including differential analysis, visualization, gene enrichment, and network modeling. They are located on the second floor of the Medical School.
CAIPP Bioinformatics and Modeling Core Team:
Dr. Rona Scott
Director of CAIPP Bioinformatics and Modeling Core
Rona.Scott@lsuhs.edu
Jian Wang, PhD
Assistant Director of CAIPP Bioinformatics and Modeling Core
Jian.Wang@lsuhs.edu
Policies
Policies:
Raw FASTQ files will be provided to the investigator via Illumina's BaseSpace website. Data generated in the Genomics Research Core Facility is strored for 3 years.
The RCF Genomics Core is not responsible for costs associated with samples that fail quality control or library preparation for services, if the failure is due to sample quality issues. This includes sample purity and sample integrity. Samples that fail quality control measures will be billed for the cost of the reagents used in these procedures. These measures are necessary, as the RCF needs to recoup the associated consumable costs for these procedures.
Samples will be put in the queue for processing ONLY after they have been received and pass all quality control measures.
Illumina Contact Information
Contact Us
Because of the cost, complexity of the process, and pros and cons of each option, careful thought should be given to the experimental design for your specific research goal. Please contact us for a consultation to ensure optimal experimental design and setup.
Camille Cannon, MS
Research Specialist, Genomics
camille.cannon@lsuhs.edu
(318) 675-4174
Rona Scott, PhD
Director, CMTV Genomics Core
Professor, Department of Microbiology and Immunology
rona.scott@lsuhs.edu
(318) 675-6263