Next Generation Sequencing

Sequencing Applications

• RNA (single-cell RNA, mRNA, total RNA, miRNA)
• DNA (amplicon, viral, bacterial DNA, mutations)
• ChIP (Chromatin Immunoprecipitation)
• 16S Metagenomics (taxonomic classification)
• Visium Spatial Transcriptomics

• 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.

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.

Sample requirements vary depending on application.  The following may be used as a guideline; however, always consult Core staff prior to sample extraction.

*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:

1. 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.
2. Sample Submission: You must provide Genomics Core staff with a completed sample submission form at the time of sample submission.
3. 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.
4. 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.
5. 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).
6. 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.
7. 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.
8. 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.

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:

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.

For Illumina sales support, please reach out to:

Lewis Gill
Executive Territory Account Manger - West TN/LA/AR/OK
(858) 281-9096
lgill@illumina.com

Corey Reier
Senior Inside Sales Representative - Mid-South
(980) 226-7533
creier@illumina.com

For orders and technical support:

(800) 809-4566