Next Generation Sequencing

Next generation sequencing services are available to the research community through the Genomics Core. The concept behind NGS technology is similar to sequencing by capillary electrophoresis – bases of a small DNA fragment are sequentially identified from signals emitted as each fragment is re-synthesized from the template strand. NGS extends this process across millions of reactions in a massively parallel fashion, rather than being limited to a single or a few DNA fragments.
 
This advance enables rapid sequencing of large stretches of DNA base pairs spanning entire genomes, with the latest instruments capable of producing hundreds of gigabases of data in a single sequencing run (“An Introduction to NGS,” 2013 - retrieved from Illumina.com). Successful Illumina sequencing is not only dependent on the quality of the DNA or RNA submitted, but also on the quality of the library created and in the selection of the appropriate protocol. Our highly experienced specialists consistently produce uniform libraries for optimal cluster generation and maximal data output.  

Fees

Fees for Core Preparation of Libraries
Application On Campus Off Campus
Illumina TruSeq Stranded Total RNA    
Sample QC, Library Preparation and Sequencing $250 $400
Data Storage (up to 1TB of data) $100 $150
Data Analysis $300 $400
Illumina TruSeq Stranded mRNA    
Sample QC, Library Preparation and Sequencing $200 $375
Data Storage (up to 1TB of data) $100 $150
Data Analysis** $300 $400
Illumina Nextera XT (DNA Sequencing)    
Sample QC, Library Preparation and Sequencing $125 $250
Data Storage (up to 1TB of data) $100 $150
Metagenomics/Amplicon    
Sample QC, Library Preparation and Sequencing $50 $100
Data Storage (up to 1TB of data) $40 $80

 

Fees for Submission of User-Prepared Libraries
Service On Campus Off Campus*
Agilent TapeStation QC and Qubit Quantification $10 $20
qPCR for Library Quantification $10 $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

 

  • Off campus fees apply to academic institutions only.  Non-academic facilities should contact the Core for additional information.
  • The Core is currently only able to return lists of differentially expressed transcripts for RNA-Seq applications.
  • 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 - Effective April 3, 2109 (shipping costs are not reflected)
Sequencing Kit Catalog Number Cycle Number Cost
MiSeq Reagent Kit v2 MS-102-2001 50 $840
MiSeq Reagent Kit v2 MS-102-2002 300 $1,080
MiSeq Reagent Kit v2 MS-102-2003 500 $1,210
MiSeq Reagent Kit v3 MS-102-3001 150 $930
MiSeq Reagent Kit v3 MS-102-3003 600 $1,580
NextSeq 500/550 Mid Output Kit v2.5 20024904 150 $1,110
NextSeq 500/550 Mid Output Kit v2.5 20024905 300 $1,790
NextSeq 500/550 High Output Kit v2.5 20024906 75 $1,525
NextSeq 500/550 High Output Kit v2.5 20024907 150 $2,920
NextSeq 500/550 High Output Kit v2.5 20024908 300 $4,680

 

Sample Coverage:  To determine the number of samples that can be pooled in one sequencing run, access the Illumina Sequencing Coverage Calculator through the link below.  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

1 x 50 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

Illumina NextSeq 550

The NextSeq 550 is a more flexible system, enabling the analysis of transcriptomes, exomes and whole genomes. It is capable of generating up to 120 Gb of output with up to 400 M single sequencing reads and up to 2 x 150 bp read lengths.  The NextSeq 550 also includes the capability of scanning cytogenomic BeadChip arrays.

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

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.

Illumina Consumable Price List - Effective April 3, 2109 (shipping costs are not reflected)Sequencing KitCatalog NumberCycle NumberCost

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.
  • 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 TruSeq Stranded mRNA, Illumina TruSeq 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 or an Illumina NextSeq 550.
  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 receive your data on a USB flash drive, external hard drive or through DropBox and will be given access to the run through BaseSpace.
  8. Bioinformatics: Once your data is generated, the Genomics Core Facility is happy to assist you with analysis for certain applications, such as RNA-Seq and variant detection.  We will advise you regarding options for analysis during the initial project consultation.  You may also contact the LSUS Bioinformatics Core.

Samples will be put in the queue for processing ONLY after they have been received and pass all quality control measures.

Data Analysis

For RNA-Seq applications, the Core is able to provide lists of differentially expressed transcripts. Our pipeline involves the following steps:

  • STAR alignment
  • RSEM counting
  • EBSeq differential expression
  • Transcript filtering with a FDR of 0.05, unless otherwise specified

For all applications, the sequence reads (in fastq format) and alignment files, if applicable, will be delivered to the investigator on a flash drive or through BaseSpace and will be available for download through Illumina BaseSpace online.

Upon request, the LSUS bioinformatics team can provide help for a deeper analysis of the sequencing data.  They are located in the Laboratory for Advanced Biomedical Informatics at LSUS.  Their website can be accessed at www.labi.lsus.edu

LSUS Bioinformatics Team

Dr. Marjan Trutschl
Office: Technology Center 216
Marjan.trutschl@lsus.edu
318-797-5131 (office)
318-795-4274 (research lab)

Dr. Urška Cvek
Office: Technology Center 215
Urska.cvek@lsus.edu
318-795-4266 (office)

 

Policies

Policies:

Data will be provided to the investigator using the most efficient method of transfer and may include cloud-based access or Dropbox sharing.   The data storage fee includes sample uploading as well as back-up of data generated in the Genomics Research Core Facility for 3 years. 

The RCF Genomics Core is not responsible for costs associated with samples that fail quality control, labeling, or library preparation for services associated with Affymetrix microarray or next generation sequencing, 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.

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 Abshire, MS, MDxT-(AAB), CLS (LSBME)
Research Specialist, Genomics
cabsh2@lsuhsc.edu
(318) 675-4174
 

Rona Scott, PhD
Director, CMTV Genomics Core
Associate Professor, Department of Microbiology and Immunology
rscott1@lsuhsc.edu
(318) 675-6263