CRI’s Sequencing Facility is a shared resource committed to enhancing research capacity for CRI and UT Southwestern scientists through the use of genomics-based technologies. The facility has an Illumina NextSeq 500 desktop Next Generation Sequencing (NGS) system optimized for high throughput, high speed and long reads.
UT Southwestern also has a Next Generation Sequencing Core Facility at the McDermott Center that provides UT Southwestern scientists with a wider array of sequencing platforms in conjunction with the Bioinformatics Platform.
Introduction to Sequencing Technology
Genomics-based technologies have revolutionized the way in which scientists approach questions in basic, clinical and translational research. NGS platforms enable a wide variety of applications, revealing limitless insights about the genome, transcriptome and epigenome of any organism.
Illumina NGS technology is based on massively parallel sequencing of millions of DNA fragments using reversible terminator-based sequencing chemistry. This technology relies on the attachment of randomly fragmented DNA to a planar, optically transparent surface. Attached fragments are extended and bridge-amplified to create an ultra-high density sequencing flow cell with millions of clusters, each containing hundreds to thousands copies of the same template.
These templates are sequenced using a robust two-color or four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. High-sensitivity fluorescence detection is achieved using laser excitation and high speed scanning optics.
Standard library preparation kits offer protocols for sequencing whole genomes, mRNA, targeted regions such as whole exomes, custom-selected regions, protein-binding regions and more. In most of the protocols, the DNA is first fragmented into a library of small segments that can be uniformly and accurately sequenced in millions of parallel reactions.
The newly identified strings of bases, called reads, are then reassembled using a known reference genome as a scaffold (resequencing), or in the absence of a reference genome (de novo sequencing). The full set of aligned reads reveals the entire sequence of the DNA sample. Alternative sample preparation methods allow the same system to be used for a broader range of applications, including gene expression, small RNA discovery and protein-DNA interactions.
The sequencing facility at CRI is open to all scientists at CRI, UT Southwestern and other interested institutions. For access to the sequencing facility, please contact Xin Liu or Jian Xu at least a week before you plan to run sequencing.
All users must complete the online training that Illumina provides. The required courses, listed below, will help familiarize you with Illumina sequencing technology and workflows.
- Sequencing: Illumina Technology.
- NextSeq 500: System Overview.
- NextSeq 500: How to Start a Run.
- NextSeq 500: Preparing Runs with BaseSpace.
- NextSeq 500: Does My Run Look Good?
- BaseSpace: Introduction.
- BaseSpace: FAQs.
Once you are an approved user, please complete the following steps.
Prepare sequencing libraries.
We suggest you pool the bar-coded samples if you are using multiplexed libraries. For help with this, please see the workflow overview below.
- Prepare samples (RNA, ChIP DNA, genomic DNA, etc.)
- Choose a library preparation kit.
- Prepare the library.
- Complete library QC.
- Library pooling (if using multiplexed library).
- Choose a library preparation kit. You can obtain library prep kits from several vendors (Illumina, NEB and NuGEN).
Once you have decided which kit to use for library prep, please follow the protocols the manufacturer provides. Pay attention to the minimum amount of DNA or RNA required for each kit and whether the provided indexing (or bar-coded adaptor) system is compatible with Illumina sequencing technology.
Check the quality of your samples
Correct sample quantification is extremely important. We highly recommend the following procedure obtain an accurate library quantification and quality check:
- Take a BioAnalyzer (or TapeStation) trace of each library to find the average library size.
- Provide these BioAnalyzer traces for review before sequencing.
- Quantify each library using Qubit at least twice.
- Dilute each library down to 10 nM using the average concentration from step 3.
- Ensure that libraries are at 10 nM using Qubit.
- Use the following formula for calculating molarity of your library: ((Concentration in ng/uL)/(660g/mol x average base pair of library)) x 10^6 = nM
- Complete the Library QC Form, and email it to Xin Liu or Jian Xu, along with the Sample Drop-off Form.
- Qubit and qPCR quantification should be largely consistent. If they are not, we suggest using the higher concentration to avoid potential over-clustering, which may significantly affect the data quality and yield. Please double-check your pooled library before sending it to us to avoid unintentional errors during sample pooling.
Provide pooled library and complete the necessary forms.
Provide pooled library as the following: 10 nM, 20 ul is recommended but the minimum amount is 4 nM, 20 ul.
Submit the pooled library.
Send the pooled library to Xin Liu or Jian Xu. We will set up the sequencing run following the workflow below:
- Select NextSeq500 Sequencing Kit (Available kits and kit overview).
- Set up runs in BaseSpace or as stand-alone mode.
- Denature and dilute libraries.
- Start NextSeq500 sequencing run.
- Monitor NextSeq500 run.
- Data analysis.
Please note the minimum sequencing unit is one flow cell (300~400 million reads, which is equal to the reads from two lanes in an 8-lane flow cell used in most facilities). We do not accept libraries with 5′ inline bar codes, because Illumina sequencing is sensitive to constant sequence within the first few bases of the read. We use Illumina NextSeq 500 High Output kits (#FC-404-2002; FC-404-2004; FC-404-2005) and Medium Output kits (#FC-404-2001; FC-404-2003) to perform sequencing. Please make sure that your library preparation method is compatible with Illumina sequencing primers.
The Illumina NextSeq 500 is the desktop NGS system optimized for speed and long reads. Each cartridge (or flow cell) produces up to 400 million reads with read lengths between 75 bp and 150 bp by either single (SR) or paired end (PE) reads to generate up to 120 Gb of data and more than 75 percent of high-quality reads (Q30). Multiple samples can be multiplexed onto a single cartridge to more efficiently distribute the reads. The turnaround time can be as fast as 30 hours per cartridge.
The NextSeq 500 is ideal for:
- Midsized project with a handful of samples.
- Transcriptome RNA-seq.
- Exome sequencing.
- Targeted sequencing.
- Amplicon sequencing.
- De novo sequencing.
Available Sequencing Types:
- 1 x 75bp (SR75).
- 1 x 150bp (SR150).
- 2 x 75bp (PE75).
- 2 x 150bp (PE150).