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 and Targeted Sequencing. If you cannot 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 be further 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 your input and projected goals. Please 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

Whole genome sequencing 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. Usually, 300 bp or 500 bp DNA-seq libraries are made and sequenced on Illumina HiSeq platforms to produce millions of reads that can be mapped to the reference genome, allowing the identification of SNVs, indels, SVs and CNVs.


De novo whole genome sequencing

De novo whole genome sequencing is used to assemble a novel genome. The method of assembly dictates the types and constuction 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 library prep for sequencing on PacBio sequencers. Please verify the library construction requirements for the assember of choice before submitting samples, as many assemblers have specific requirements for the construction of the libraries providing sequence. For example, ALLPATHS requires data from two distinct library types made from the same sample: both a mate pair library with a specific insert size and a standard library with a specific insert size.

To construct mate pair libraries, we use Illumina's Nextera Mate Pair kit. We can generate 3-5 kb, 5-7 kb, or 7-10 kb 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 requirement 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

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 percent 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 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. Our standard capture is the NimbleGen SeqCap EZ Exome Library v3.0 with a capture size of 64 Mb.


Custom targeted sequencing

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 NimbleGen SeqCap EZ Library captures. A custom capture panel needs to be designed and ordered directly from Roche.
  2. For amplicon-based capture, we offer Illumina TruSeq Custom Amplicon and 16S Metagenomics.
  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 longer than 300 bp.

Whole Genomic bisulfate sequencing (WGBS)

WBGS can allow detection of 5mc, 5hmc and/or 5fc methylation across a genome.


Sequencing of chip/hic/other dna

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.


restricted site associated dna sequencing (rad-seq)

RAD-Seq is a reduced-representation sequencing method typically used to identify single nucleotide polymorphisms (SNPs) distributed randomly across the genome. RAD tags, which are the DNA sequences that immediately flank each instance of a particular restriction site of a restriction enzyme throughout the genome, are isolated, made into a sequencing library by litigation of sequencing adapters, and sequenced on a high throughput Illumina sequencer. RAD-Seq has been used for association mapping, QTL-mapping, population genetics, ecological genetics and evolution, and phylogenetics.

*For any samples that do not meet our input specifications, or cannot be quality controlled due to limited DNA amounts or volume, we cannot warranty any technical aspects or analysis performance. For ChIP-seq samples and all amplicons, we do not guarantee the results because we have no control over the highly complex nature of the ChIP portion of a ChIP-seq experiment or over the DNA content of an amplicon pool.  If we are unable to quality control the submitted DNA, it is impossible to predict the performance of an experiment, i.e we can't guarantee a high-quality sequencing library will be produced nor can we guarantee that the results will show expected, high quality data.


related links:

Genomic Analysis and Bioinformatics Shared Resource