The Genomics Laboratory

Next Generation Sequencing using Illumina systems, that produce short reads of up to 300bp from each end of a DNA fragment, remains the gold standard for many avenues of genomic research, particularly RNA-Seq or identification of SNPs or small indels in genomic sequences. If you are working on an organism that already has a high quality genome sequence, then it is likely that this will be the technique that you will use for your experiments. If you work on species whose genome sequence has yet to be determined, you might also want to combine this with longer read, nanopore sequencing.

The genomics lab can provide end-to-end support, from experimental design through to analysis (working with members of our data science team) for all your illumina sequencing needs. Our talented technicians provide a full service option for Illumina library preparation, working from a whole host of possible sample types, including, but not limited to:

• Whole (shotgun) genome or metagenome DNA sequencing or resequencing
• RNA Sequencing, including polyA mRNA or rRNA-depleted total RNA for whole transcriptome studies, miRNA, or RNA-IP
• ChIP-Seq
• ATAC-Seq
• Targetted amplicon sequencing (eg. 16S amplicon for metagenomic profiling)
• Target enrichment 

While we do not have Illumina sequencers in house, we work closely with neighbouring genomics facilities and sequencing suppliers to secure the best quality and most cost-efficient sequencing for our users. Please contact us to for more information on how we can tailor our services to suit your project needs.

The Genomics lab are proud and experienced service providers of Oxford Nanopore Technologies' sequencing, using both the MinION and PromethION systems.   

Nanopore sequencing offers a unique technology to enable direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids (either DNA or RNA samples) are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA or RNA sequence. This allows for both considerably longer reads than those offered by conventional Sanger and Illumina sequencing-by-synthesis approaches, and also allows detection of modifications on the nucleic acids. Standard workflows now exist that allow us to call presence of 5mC and 6mA on native DNA with confidence.

The longer reads that nanopore sequencing can produce have revolutionised the analysis of genomes by allowing us to sequence and assemble those parts of the genome, such as longer repeat sequences, that are intractable using short-read technologies. They can also be used to generate full-length cDNA sequences, producing excellent reference transcriptomes for novel species, providing vital insights into alternative splicing or identifying fusion transcrips.

We have extensive experience working with a broad spectrum of sample types and species, from microorganisms and viral phages, to plants, fungal samples, complex metagenomes, mammalian samples and clinical tissues, across a wide range of Nanopore sequencing applications, including (but not limited to):

• Novel genome assembly
• Genome resequencing for structural variant calling
• Whole metagenome analysis and assembly
• Full length transcript analysis for splice variants
• Novel organism transcriptome assembly for genome annotation
• Methylation calling with eukaryotic genomes

While nanopore sequencing may not provide as high base accuracy as some traditional sequencing approaches, their methods are rapidly evolving, and have improved immensely since their inception. Our methods of working adapt accordingly, allowing us to provide our users with the most up-to-date, accurate and high yielding experiments possible. 

Please contact us to discuss your interest in nanopore sequencing and the services we can provide for you.

Close collaborations between the Imaging & Cytometry, Genomics and Data Science labs allow our facility to support state-of-the-art single cell isolation, processing and analysis, whether this be multiwell-plate based analysis of flow-sorted cells, or through the use of our 10X Genomics Chromium platform.

Services offered include, but are not limited to:

• Gene expression analysis (3’, 5’ or full length mRNA sequencing)
• Feature barcoding
• Immune cell profiling
• ATAC-Seq (Assay for Transposase-Accessible Chromatin)
• Multi-omics (combined 3’ gene expression and ATAC-Seq analysis)

We can support our users through every stage of the process, from experimental design, through single cell isolation and sample prep, quality control and data analysis. Please contact us for more information or to discuss potential projects.

The Genomics, Imaging and Cytometry and Data Science labs work together to support a range of spatial analysis platforms as full service or collaborative projects, using both 10X Genomics and Nanostring systems. Please contact members of the Genomics (sally.james@york.ac.uk) or Imaging (grant.calder@york.ac.uk) teams for further information and discussion.

• GeoMx Digital Spatial Profiler (DSP)

 Whole tissue sections (FFPE & fresh frozen) or TMAs can be probed for either for protein (antibodies up to 100 plex) or RNA (oligonucleotides up to 20,000 plex) targets. NanoString’s unique chemistry invisibly tags probe molecule with an indexing barcode oligonucleotide that can be released upon exposure to UV light. Using standard immune or in situ labelling protocols, GeoMx panels (probe cocktail) combined with up to 4 fluorescent morphology markers can simultaneously probe the tissue. The staining patterns of morphology markers are then used to guide the selection of region of interests (ROIs), where targeted UV light releases “indexing barcodes” allowing their extraction. To reveal which probes where bound to target region, the released barcodes are later identified and counted using either nCounter (low plex) or NGS (high plex) platforms. Due to the GeoMx DSP flexible UV targeting system, illumination patterns can range from simple geometric shapes or be subdivided into complex patterns e.g. tumour or rare cells, using intensity based segmentation of the morphological markers. This allows for enrichment of target cells giving robust results. Although, it is technically possible to select a single cell the statistical noise would render such data unusable. Thus NanoString recommend collecting minimum of 20 cells for protein probes and 100 cells for RNA probes per ROI.

GeoMx protein panels are modular using validated antibody probes covering immunology, oncology and neuroscience with an expanding portfolio and the possibility of adding up to 10 custom targets. The GeoMx RNA panels provide a spatial view of thousands of protein encoding genes with possibility of adding hundreds of custom targets. Combine GeoMx Protein and RNA -NGS panels on a single slide to get a proteogenomic view of your sample. Explore other species by making GeoMx custom RNA panels of up to 400 targets.

• 10XGenomics Visium

Map the whole transciptome across a tissue (FFPE or Frozen) using Visium Spatial capture slide with ~5000 evenly spaced (100um centre to centre) capture spots (55um diameter) covering on average 1 to 10 cells depending on tissue type. Tissue sections can be stain using either H&E or fluorescent markers and imaged to give morphological context. Further on slide processing steps are used to permeabilise tissue releasing mRNA (fresh frozen) or ligated probes (FFPE) that are captured on the slide. Spatial barcodes for each capture spot are added via a cDNA extension reaction. The barcoded target molecules can be pooled to generate a sequencing library and sequenced using Illumina technology giving a transcriptional profile for the whole tissue. The unique capture spot barcode allows targets to be spatially assigned giving an unbiased transcriptome view across the tissue. 

Real-time PCR provides the ability to monitor the accummulation of fluorescent products throughout the PCR procedure.  Reactions are characterised by the cycle number (Ct) at which products are first detected, rather than the amount of target accumulated after a fixed number of cycles. The more of a specific target nucleic acid is present in a sample, the sooner a significant increase is detected and the lower the Ct value which is measured. This allows for both absolute or relative quantitation of nucleic acids over many orders of magnitude.

The genomics lab provides access and training to our users in the use of qPCR. We maintain and run three qPCR instruments:

• StepOnePlus (Applied Biosystems) 
• QuantStudio 3 (96 well, 0.1 ml FAST block capacity)
• QuantStudio 7 Pro (Interchangable 0.1 ml FAST 96 well and 384 well blocks)

Two types of chemistry can be used:

• SYBR Green - Double-stranded DNA-binding dye allows measurement of the accumulation of PCR products
• TaqMan - Fluorogenic probes for detection of specific PCR products, allowing allelic discrimination.

All instruments can also be used for high-resolution melting studies.

Agilent 2100 Bioanalyser and 4200 Tapestation

The Agilent 2100 Bioanalyzer and 4200 Tapestation systems provide rapid analysis of the quality and quantity of valuable RNA and DNA preparations. As little as 200pg of total RNA, 500 pg of mRNA or 5pg of DNA can be analysed in as quickly as 30 minutes. The Bioanalyzer is a chip-based capillary electrophoresis system and replaces formaldehyde / agarose gels. The system can be used with RNA fragments up to ~6kb and DNA up to ~12 kbp. Higher numbers of samples, or analysis of larger DNA fragments  can be run on an Agilent TapeStation 4200 system, a fully automated miniaturised electrophoresis system. The system can produce an integrity number (RIN for RNA, DIN for DNA) which is a good measure of the integrity of the sample. Samples are scored between 1 (poor quality, degraded material) and 10 (good).

Further information on what the machines can do can be found at Agilent.

Please contact our staff if you are interested in running samples using these systems; we provide a full service and can assist with running the samples on the appropriate system to suit your needs.

Nanodrop Spectrophotometers

No need for cuvettes (although these can be run using the ND1C system); the nanodrop instruments use surface tension to form a column of liquid across a 1mm gap between optical fibres, measuring a full spectrum of wavelengths as required, scanning between 220 and 750 nm,

1µl minimum sample size (1.2µl for the eight channel machine) - Allows analysis of precious samples without dilution

Applications include:

• Nucleic acid concentration and purity measurements (concentration range 2ng/µl - 3700ng/µl).
• Microarray sample analysis: Determine dye label concentration of hybridisation samples.
• Absorption of fluorescent Cy and Alexa dyes measured down to 0.2 pmole/µl
• Protein assays: Bradford and BCA
• Cell density measurement
• General UV-Vis spectrophotometer

More details on the NanoDrop Products website.

If you would like to use, or see it demonstrated, please contact us.