- Nucleic acid extraction and library construction services include FFPE genome, PCR-free genome, low input DNA, bisulphite, chromatin immunoprecipitation (ChIP), mRNA (strand specific), ribodepletion, miRNA, exome and custom capture.
- Sequencing services include whole genome, whole transcriptome (ribosomal depleted, or polyA RNA), epigenome, ChIP, exome capture (exons only, or UTR included), miRNA, long reads and linked read as well as customized sequencing.
Whole genome sequencing is 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.
- Genomic DNA is ligated to adapters without amplification creating the best quality and least biased whole genome.
- We specialize in human sequencing but can sequence any species from bacteria to mammalian and large plant genomes.
- Various genome coverages are available—30-40X is recommended for human germline analysis and 80X for somatic analysis.
Formalin-fixed paraffin-embedded (FFPE)
- We have optimised plate-based FFPE nucleic acid extraction and library construction, these libraries are amplified and have shorter inserts due to sample quality.
- Due to the variable quality of FFPE samples, this sample type routinely requires more sequences to be generated to reach the same coverage levels as non-FFPE samples.
- We also offer amplified whole genome sequencing, in particular for limiting samples, eg., circulating DNA or small biopsies.
Whole transcriptome RNA sequencing is a next generation sequencing technique that measures the abundance of RNA transcripts and the presence of mutations or fusion transcripts in a sample. It is a powerful tool for understanding dynamics in the transcriptome, including gene expression level differences between different physiologic conditions or changes that occur during development or over the course of disease progression.
Ribosomal RNA depleted
- Stranded total RNA-seq with ribosomal depletion selectively removes ribosomal RNA from total RNA samples by hybridization. A complete transcriptome profile is produced that can be utilised for expression studies, alternative splicing, novel isoforms and expressed structural rearrangements.
- This can be performed on human or mouse samples and also functions well with lower quality RNA such as those extracted from FFPE tissue samples.
PolyA+ messenger RNA (mRNA)
- Stranded mRNA-Seq is a popular tool for estimating gene expression levels and comparing differential gene expression in model organisms.
- mRNA-Seq (PolyA+ selection) can provide valuable information about alternatively spliced isoforms and can help identify novel fusion transcripts.
Micro RNA (miRNA)
- miRNA analysis provides the ability to discover, measure and compare expression levels of known miRNAs and other small non-coding RNA species.
- Using the 10x Genomics Chromium system we can provide single cell sequencing services, including 3’ polyA RNA sequencing and whole genome sequencing aimed at copy number profiling.
Epigenetics is used to describe heritable genetic modifications that are not attributable to changes in the primary DNA sequence. Epigenetic modifications play a crucial role in gene expression, and thereby underpin the development, regulation and maintenance of the normal cell. Lifestyle, nutrition and environmental factors can all lead to epigenetic changes. Two of the most commonly studied epigenetic modifications involve the binding of proteins to DNA and the methylation of cytosine (C) nucleotides in the context of a CpG dinucleotide. Because the expression of miRNAs can impact epigenetic mechanisms, they can also contribute to epigenetic changes.
Chromatin immunoprecipitation (ChIP)
- ChIP is a powerful experimental approach enabling the identification of proteins associated with specific regions of the genome.
- Histone modifications can impact gene expression by altering chromatin structure. Histone H3 modifications include methylation of lysine residues 4 (H3K4me1 and H3K4me3), 9 (H3K9me3), 27 (H3K27me3) and 36 (H3K36me3) and acetylation of lysine residue 27 (H3K27ac). Quantitative detection of these histone ‘marks’ provides useful information on the epigenetic regulation of cellular processes.
- We recommend sequencing the narrow, or punctate, marks (H3K4me3 and H3K27ac) with 50 million reads and the broad marks (H3K4me1, H3K9me3, H3K27me3, H3K36me3) and input chromatin control with 100 million reads.
Whole genome bisulphite (WGBS)
- WGBS is a sequencing technology used to determine the DNA methylation status of individual cytosines by treating the DNA with sodium bisulphite before sequencing. The chemical compound sodium bisulphite converts unmethylated cytosines into uracil. The cytosines that have not converted to uracil are methylated. After sequencing, the unmethylated cytosines are read as thymines.
- A sequencing depth of 30X is recommended.
- Methyl capture is a targeted approach to bisulfite sequencing.
- We target over 3.3 million CpG dinucleotides, interrogating biologically important methylome targets using hybridization oligonucleotide probes. This approach supports both screening and biomarker discovery in the methylome.
Direct detection of methylcytosine via long-read sequencing
- Using nanopore sequencing, we can directly identify DNA and RNA base modifications at nucleotide resolution.
- Compared to whole-genome bisulphite sequencing, long-read technology calls a higher number of CpG positions in the genome, requires less sequencing data, and shows more even genomic coverage with considerably lower GC bias; analysis runtime is also significantly shorter.
- HiC is a proximity ligation method that captures the three-dimensional (3D) organizational structure of chromatin, where genomic sequences that are distal to each other in linear distance can be closer to each other in the 3D space. The high-resolution, genome-wide map of interacting genetic loci that is generated from Hi-C data can then be used across multiple genomic applications including identification of promoter-enhancer interactions for gene regulation studies and scaffolding contigs for genome assemblies to define chromosomes de novo.
- We have optimized workflows for different tissues as well as isolated nuclei.
Whole exome sequencing (WES) consists of sequencing only a specific subset of the genome, the exons, which represent the entire protein coding part of the genome. WES can be used to study genetic variations involved in inherited as well as in sporadic disorders, including cancers, and provide an alternative to whole genome sequencing. We offer two types of exome capture protocols as well as custom gene/feature panels.
- Covering 39 Mb of the human genome, representing the coding exons of 19,396 genes, we offer the xGen Exome Research Panel (v1.0) from Integrated DNA Technologies.
Exons plus UTRs
- Covering 89 Mb of the human genome, including coding exons and 5’ and 3’ untranslated regions (UTRs), we offer the SureSelect Human All Exon (V6+UTR) exome from Agilent.
- We can also provide custom gene/feature panels.
- Please contact us to discuss your project requirements.
Custom projects and additional services are available upon request, including submission of constructed libraries. Please connect with us to explore the possibilities.
- We accept constructed libraries.
- For information about what we accept and our requirements please check our Library Construction and Sequencing FAQ and our Constructed Library Submission Requirements document.
- Please contact us if you have questions.
- Please contact us to discuss your specific research needs and we can advise on the best technology and approach for your project.
The NextSeq 2000 is available to researchers in a walk-up sequencing mode. Users will bring their own ready to sequence library pools and the appropriate sequencing kit (available directly from Illumina), once loaded, data will be streamed directly to a user’s Illumina BaseSpace account for immediate access.
This service allows direct access to sequencing infrastructure and allows rapid technical development testing as well as fast turnaround for BC Cancer and other researchers. At launch there will be no additional charge for access to this sequencer.
The NextSeq 2000 is a highly flexible platform generating up to 400 Gb sequence data. There are currently three sequencing flowcells (P1, P2, and P3) of variable read lengths from 50 bases to 2x300 bases and 200 Million to 2.4 Billion individual reads in run times of 11 to 48 hours. Follow this hyperlink for detailed specifications.
Following an initial training and safety orientation at the GSC Echelon site users will be able to schedule use of the sequencer through an online booking portal.
For access and any sequencing enquiries please contact our Collaborative Services team (firstname.lastname@example.org).