The HTSF is a full-service sequencing facility. We offer multiple sequencing platforms to assist with your genetic and genomic research. Our services also include using state-of-the-art techniques and data analyses.

DNA Sequencing

Complete genome re-sequencing – DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions (i.e. clusters of genes or operons), full chromosomes or entire genomes, of any organism. We routinely perform comprehensive polymorphism and mutation discovery in individual genomes.

  • This service is most commonly performed using Illumina HiSeq400 paired end 2 x150 base sequencing.
  • If there are substantial gaps in the sequence assembly, other platforms such as Oxford NanoPore MinION or Pacific Biosciences Sequel can frequently be used to create larger contiguous assemblies to supplement the deep coverage from Illumina sequence runs.

ChIP-seq – ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest.

  • This is typically performed on HiSeq4000 using paired end 2×50 base sequencing.
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FAIRE-Seq (Formaldehyde-Assisted Isolation of Regulatory Elements) is a method used for determining the sequences of those DNA regions in the genome associated with regulatory activity. In contrast to DNase-Seq, the FAIRE-Seq protocol doesn’t require the permeabilization of cells or isolation of nuclei, and can analyze any cell types. In a study of seven diverse human cell types, DNase-seq and FAIRE-seq produced strong cross-validation, with each cell type having 1-2% of the human genome as open chromatin.

  • This is typically performed on HiSeq4000 using paired end 2×50 base sequencing.
Bisulfite sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA to determine its pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity. Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Therefore, DNA that has been treated with bisulfite retains only methylated cytosines. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. The objective of this analysis is therefore reduced to differentiating between single nucleotide polymorphisms (cytosines and thymidine) resulting from bisulfite conversion.

  • This is typically performed on a HiSeq2500 Rapid Run protocol with paired end 2×100 base sequencing.
Metagenomics is the study of genetic material recovered directly from environmental samples. The broad field may also be referred to as environmental genomics, ecogenomics or community genomics. While traditional microbiology and microbial genome sequencing and genomics rely upon cultivated clonal cultures, early environmental gene sequencing cloned specific genes (typically the 16S rRNA gene) to produce a profile of diversity in a natural sample. Such work revealed that the vast majority of microbial biodiversity had been missed by cultivation-based methods. Recent studies use either “shotgun” or PCR directed sequencing to get largely unbiased samples of all genes from all the members of the sampled communities. Because of its ability to reveal the previously hidden diversity of microscopic life, metagenomics offers a powerful lens for viewing the microbial world that has the potential to revolutionize understanding of the entire living world. In medical research discovery of infectious and commensal flora relies increasingly on metagenomics analyses.

  • This is most commonly performed on a MiSeq with a paired end 2×300 base sequencing protocol.
This is a high throughput system capable of analysis of single cell genetic differences within a cell population. This system can be used to provides long range information on a genome-wide scale, including variant calling, phasing and extensive characterization of genomic structure. It can also use the power of linked-reads to fully resolve genic phasing, structural variation, and detect variants in previously inaccessible and complex regions of the exome.

  • This is typically performed on HiSeq4000 using paired end 2×150 base sequencing.
An efficient and high-throughput technique used to analyze the genome-wide methylation profiles on a single nucleotide level. This technique combines restriction enzymes and bisulfite sequencing in order to enrich for the areas of the genome that have a high CpG content. Due to the high cost and depth of sequencing needed to analyze methylation status in the entire genome, Meissner et al. developed this technique in 2005 in order to reduce the amount of nucleotides needed to be sequenced to 1% of the genome. The fragments that comprise the reduced genome still include the majority of promoters, as well as regions such as repeated sequences that are difficult to profile using conventional bisulfite sequencing approaches.

  • This is typically performed on a HiSeq2500 Rapid Run protocol with paired end 2×100 base sequencing.
Restriction site associated DNA (RAD) markers are a type of genetic marker which are useful for association mapping, QTL-mapping, population genetics, ecological genetics and evolution. The use of RAD markers for genetic mapping is often called RAD mapping. An important aspect of RAD markers and mapping is the process of isolating RAD tags, which are the DNA sequences that immediately flank each instance of a particular restriction site of a restriction enzyme throughout the genome. Once RAD tags have been isolated, they can be used to identify and genotype DNA sequence polymorphisms mainly in form of single nucleotide polymorphisms (SNPs). Polymorphisms that are identified and genotyped by isolating and analyzing RAD tags are referred to as RAD markers.

  • This is typically performed on HiSeq4000 using single end 50 or 100 base sequencing.

RNA Sequencing

RNA-Seq (RNA sequencing), also called whole transcriptome shotgun sequencing (WTSS), uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment in time. RNA-Seq is used to analyze the continually changing cellular transcriptome. Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5′ and 3′ gene boundaries. There are a variety of approaches to transcriptome sequencing, the best approach will depend on your needs. Sequencing reactions can be strand specific if desired which allows differentiation of overlapping transcripts on opposite strands. Discovery of novel noncoding regulatory RNAs can also be performed.

  • This is typically performed on HiSeq4000 using paired end 2×50 base sequencing
MicroRNA sequencing (miRNA-seq), a type of RNA-Seq, is the use of next-generation sequencing to sequence microRNAs, also called miRNAs. miRNA-seq differs from other forms of RNA-seq in that input material is typically enriched for small RNAs. miRNA-seq allows researchers to examine tissue-specific expression patterns, disease associations, and isoforms of miRNAs, and to discover previously uncharacterized miRNAs. Evidence that dysregulated miRNAs play a role in diseases such as cancer has positioned miRNA-seq to potentially become an important tool in the future for diagnostics and prognostics as costs continue to decrease. Like other miRNA profiling technologies, miRNA-Seq has both advantages (sequence-independence, coverage) and disadvantages (high cost, infrastructure requirements, run length, and potential artifacts).

  • This is typically performed on HiSeq4000 using single end 50 base sequencing.
This is a high throughput system capable of analysis of single cell discrimination of genetics differences within a cell population. Tools include single cell 3’ expression measurement and single cell V(D)J transcript analysis.

  • This is typically performed on a HiSeq2500 Rapid Run protocol. Most commonly with paired end 2×100 base sequencing but check with the lab since this could change depending on your requirements.
HTG Molecular’s EdgeSeq is a fully automated sample and library preparation platform for targeted RNA sequencing that pairs HTG’s extraction-free,high-specificity Edge Chemistry with the high sensitivity and dynamic range of next-gen sequencing. EdgeSeq enables digital quantitation of miRNA and mRNA expression from difficult sample types such as formalin-fixed, paraffin-embedded (FFPE) tissues, plasma and exosomes.

  • This is typically performed on a HiSeq2500 Rapid Run protocol with single end 50 base sequencing.
Microarrays are a mature technology used to detect known sequences and to read out relative abundances, not absolute values. All methods use fluoresce Cy-dye labeling and long oligo (60 mer) Agilent high density microarrays. These are used typically for RNA expression analysis. Unlike early generation arrays, these are run as single color arrays (Cy3 only) and use a labeled sample or reference hybridized on a separate microarray and a digital comparison is performed. All samples are amplified, so very small sample sizes can be accommodated. Microarrays can also be used for analysis of difficult samples suggest FFPE RNA.

Bioinformatics & Analysis

Please contact Hemant Kelkar for specific information about hardware or software resources available on campus for the analysis of NGS data.

FAQs

Have a question? Check out our list of frequently asked questions