SGT Services

The Sequencing and Genomic Technology shared resource provides DNA sequencing and RNA sequencing services as well as nucleic acid extraction and single cell services. 

DNA Sequencing

The Sequencing and Genomic Technology shared resource provides a large array of DNA sequencing services, such as Whole Genome Sequencing, Whole Exome Sequencing or Targeted sequencing. If you do not find the DNA sequencing service you need for your project in the list below, please contact us.

Upon delivery to our facility, all DNA samples are evaluated for concentration by Qubit®. DNA submitted for PacBio sequencing will further be run on TapeStation or Fragment Analyzer to check integrity of the DNA. Before submitting samples, please review our sample submission requirements.

Choosing the correct DNA sequencing service for your samples depends on the organism, total DNA available for input, and project goals. Contact us with questions regarding experimental design or DNA service options. Most next generation sequencing projects require special consideration. We recommend contacting us before initiating a project with us.

All of the services listed below are compatible with current Illumina sequencing platforms. The adequate sequencing platform for your project will depend on sequencing depth (sequencing throughput) and read length required for your project. PacBio sequencers can also be used for amplicons sequencing and whole genome sequencing either in combination with Illumina sequencing or by itself.

Services

Whole genome sequencing can be used for interrogating single-nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), and copy number variants (CNVs) in coding and non-coding regions of the genome. 500bp DNA-seq libraries are usually made and sequenced on Illumina platforms to produced millions of reads that can be mapped to the reference genome allowing the identification of SNVs, indels, etc. For small genomes like bacterial genomes,  sequencing can also be done on PacBio sequencers. The technology is particularly useful if you are looking for large structural variation.

For assembly of a novel genome. The method of assembly dictates the types and construction of libraries for sequencing. We offer standard DNA-seq libraries with different insert/fragment sizes and mate pair libraries. We also offer PacBio large insert size libraries prep for sequencing on PacBio sequencers.  Please verify the library construction requirements for the assembler of choice before submitting samples, as many assemblers have specific requirements for the construction of the libraries providing sequence.

To contruct mate pair libraries, we use Illumina's Nextera Mate Pair kit. We can generate 3-5kb, 5-7kb, or 7-10kb libraries. Mate pair libraries may be sequenced on all Illumina sequencers.

To construct standard DNA-seq libraries, we use Kapa Hyper prep kits. Insert size can be tweaked to accommodate the requirements of the assembler that will be used to assemble the data.

To construct PacBio libraries, we use the PacBio large insert library prep kit.

Whole-exome sequencing refers to sequencing all of the protein-coding genes in a genome. The exome represents less than two percent of the human genome but contains about 85% of known disease-causing variants. Whole-exome sequencing allows for the identification of genetic variation that is responsible for both mendelian and common diseases without the high costs associated with whole-genome sequencing.


Whole-exome sequencing consists of preparing a genomic DNA library and selecting only the subset of the library that encodes the protein coding genes by in-solution enrichment (hybridization) of the library to biotinylated oligonucleotide capture probes and immobilization on streptavidin-coated beads. The captured DNA is then sequenced using Illumina high throughput DNA sequencing technology. We currently provide Whole Exome capture using either the IDT xGen® Exome Research Panel, which has a capture size of ~40Mb. We also offer capture using the Agilent SureSelect Human all Exon V7, which has a capture size of 48.2Mb. We can also provide whole exome services for mouse, rat, zebrafish using Agilent SureSelect kits or other vendors.

Custom targeted sequencing is designed to isolate and deep-sequence a specific region of the genome. Targeted sequencing includes methods such as gene panels, cancer panels, or amplicon sequencing where regions or genes of interest are amplified and sequenced.

Whether you are interested in a few genes, or a few thousand genes, we can design a gene panel that assays your genes of interest with high sensitivity and specificity. Specific genes or mutations that have established relevancy to a particular cancer phenotype can be sequenced using available cancer panels. Other regions of interest such as the hypervariable regions of the microbial 16S rRNA gene, used to determine the type and relative abundance of bacterial and archaeal species in heterogeneous samples (e.g. soil, marine, or gut microbiome) can be sequenced by amplicon sequencing.

  1. For in-solution capture, we offer Agilent SureSelect captures, Roche SeqCap captures or Twist.  A Custom capture panel needs to be designed and ordered directly from those companies. We can facilitate that if you send us the list of genes you are interested in.
  2. For amplicon-based capture, please contact us. We typically work with Qiagen
  3. We will also accept any amplicon that you have amplified, and we will create a sequencing library from the DNA. Sequencing libraries can be prepared for sequencing on Illumina sequencers or PacBio sequencers if amplicon size is too long for Illumina sequencing (typically max 600bp).

Methylation sequencing can be done at the whole genome level or using a cheaper more targeted approach. We can prepare both type of libraries. For targeted Methyl-seq, we can prepare libraries using the Agilent SureSelect XT Targeted Methyl-seq panels for Human, Mouse or Rats.

We do not perform ChIP or 4C/5C/HiC protocols, but we will create a sequencing library from the DNA obtained from those protocols and sequence it to the requested specifications.

RNA Sequencing

We provide a large array of RNA sequencing services such Stranded mRNA-seq, smRNA-seq or ultra low input RNA-seq. If you do not find in the list below the RNA sequencing service you need for your project, please contact us.

Upon delivery to our facility, all RNA samples are evaluated for concentration by Qubit® and for integrity by 2100 Bioanalyzer, TapeStation or Fragment Analyzer. Before submitting samples, please review our sample submission requirements.

Choosing the correct RNA sequencing service for your samples is dependent on the organism, total RNA or number of cells available for input, and project goals. Contact us for questions regarding experimental design or RNA service options. Most next generation sequencing project requires special consideration. We recommend contacting us before initiating a project with us.

Services

This is a popular tool for estimating gene expression levels and for comparing differential gene expression in model organism. mRNA-Seq can also provide valuable information about alternatively spliced variants and can contribute to identify novel isoforms. Among many other advantages, RNA-Seq allows for highly accurate expression quantitation within a wide dynamic range. We offer complete solutions for mRNA-Seq projects including experimental design, RNA-seq library preparation, and high throughput Illumina sequencing.

We currently use the Kapa Stranded mRNA-seq library prep kit to make sequencing libraries and libraries are typically sequenced on Illumina instruments with a 50bp single end reads length. Paired-end sequencing with longer read length can also be done if one is interested in RNA-splicing.

For RNA extracted from humand blood, we are using the Nugen Universal Plus mRNA-seq kit with Globin AnyDeplete to prepare libraries.

Transcriptomes can also be assembled without the need of a reference genome. This approach is useful for determining gene sequences of non-model organisms. We offer services for performing whole transcriptome de novo sequencing on Illumina platforms and PacBio platforms. De novo transcriptome assembly using Illumina platforms requires 150bp paired end sequencing. The long reads generated by PacBio sequencing provide full-length reads spanning entire transcript isoforms from the 5' end to the polyA-tail (see PacBio Iso-Seq description). The Iso-Seq approach does not require any assembly and provides with a full, accurate, and least biased transcriptome data.

Total RNA-Seq with ribosomal reduction selectively removes ribosomal RNA from total RNA samples. In contrast to polyA+ enrichment mRNA-seq, this approach preserves non-polyadenylated RNAs to investigate broader classes of RNAs including immature mRNAs and non-polyadenylated ncRNAs. RNA-seq libarires are prepare using the Illumina TruSeq Stranded total RNA-seq kit in combination with a wide variety of ribosomal reduction kits (Illumina Ribozero kit) depending on the sample type:

  • Ribozero Gold – removes cytoplasmic and mitochondrial rRNAs from human/mouse/rat samples
  • Ribozero Globin – removes Globin mRNAs and  cytoplasmic and mitochondrial rRNAs from human/mouse/rat

Small/mi RNA analysis provides the ability to discover, measure and compare expression of know microRNAs and other small non-coding RNA. It also allows to detect novel microRNA targets. We use the Qiagen miRNA Library Prep kit  for all the miRNA library prep. 

Ultra-Low-Input RNA-Seq can be used to generate gene expression data from few or even single cells. Ultra-low-input RNA-Seq provides the opportunity to study differences between cells or cell types with an unprecedented resolution. This allows for a better understanding of biological differences between cells within a tissue/tumor and characterize subpopulation responses to environmental cues. We use the Clontech Ultra Low Input RNA SMARTer mRNA amplification Kit for the initial RNA amplification which provides a fast and simple method for preparing amplified cDNA from total RNA for RNA-Seq applications. The obtained dscDNA is then converted into a sequencing library using Kapa Hyper Prep Kit.

Studying gene expression of FFPE samples is extremely valuable for better understanding diseases. Unfortunately, FFPE samples can be significantly degraded and traditional RNA-seq library preparation methods often fail to generate optimal libraries. We can use for your FFPE sample the Illumina RNA Exome kit, an alternative sample preparation method enabling the generation of high quality RNA-Seq results from degraded samples.

Nucleic Acid Extraction Services

Nucleic acid extractions are performed on our Qiagen QIASymphony instrument or our Roche Magnapure 96 instrument.

Services

Total RNA will be extracted using the QIAsymphony PAXgene Blood RNA Kit.

All blood samples (2.5ml) should be collected in PAXgene Blood RNA Tubes. Immediately after blood collection, gently invert the PAXgene Blood RNA Tubes 8–10 times. Store the PAXgene Blood RNA Tubes upright at room temperature (18−25oC) for 2-72 hours before transferring to freezer. The PAXgene Blood RNA Tubes can be stored at −20oC and below. If tubes are to be kept at temperatures below −20oC, freeze them first at −20oC for 24 hours, then transfer them to −80oC.

Tubes should be delivered to us frozen. Note that the frozen PAXgene Blood RNA Tubes are very fragile and break easily.

  • Tubes containing material for isolation must be clearly labeled with your DUGSIM order number and sample number (“4596_S001”). 
  • For human blood samples, please make sure to provide us with your IRB number to confirm that your project has the appropriate IRB approval.

Total RNA from cell pellets will be extracted using the Qiagen QIAsymphony RNA Kit

Cell pellets should have between 1-3 million cells.

Cells should be pelleted in Sarstedt 2.0 mL tubes - cat # 72.693 or 72.608

Single Cell

We offer single-cell multi-omics assays on the Tapestri Platform. This innovative platform analyzes genotype and phenotype simultaneously from the same single cells, which lets you reveal clonal heterogeneity and target comprehensive biomarkers.