Methods Overview

This page provides a comprehensive overview of the methods available at the DcGC. It should serve as a guide for understanding the technical aspects and uses of the different technologies and workflows. For practical details on how to prepare samples, please check our guidelines page.

Bulk sequencing applications

Bulk sequencing applications are generally used for profiling tissues or cell populations, delivering generally large amounts of data at relatively lower costs.

genomics

The sequencing of the genomic sequence of an organism has valuable applications in basic and applied research, as well as in the clinical setting. Whole genome sequencing (WGS) approaches enable de novo or re-sequencing of entire genomes, while targeted sequencing approaches deliver information on specific regions of interest. At the DcGC, we offer start-to-end assays for genome sequencing using short- and long-read technologies.

Below you can fine more information about the methods offered by the DcGC for genome sequencing:

Whole Genome Sequencing

de novo sequencing

de novo whole genome sequencing is a powerful technique for a better understanding of the underlying genetic diversity among individuals and species. It is generally the preferred method of choice when the aim is producing a reference genome of a species when no assembly is available, or sequencing a highly divergent genome.

Sequencing technologies that produce longer reads (Kb or Mb lengths), such as PacBio or Oxford Nanopore, are more appropriate for this purpose because these longer stretches of information facilitate the assembly process. Among the advantages is the reduced complexity of the assembly, higher contiguity and resolution in complex genomic regions, and the ability to phase the haplotypes and detect structural variants.

Long-read genomics data is often complemented with short-read Chromosome Conformation data (HiC workflow) to increase the fidelity up to chromosome-level assemblies. These technologies are often proposed together as one package.

We have significant expertise in high quality DNA extraction and long-read sequencing technologies for whole genome sequencing and assembly. Our Long-Read Unit has sequenced and assembled the genomes of many species, including many vertebrates in the context of large-scale sequencing consortia (VGP, Bat1K), and the complex genomes of the planaria, axolotl and lungfish.

re-sequencing

Whole genome re-sequencing is relevant for variant analysis, including identification of single nucleotide polymorphisms (SNPs), insertions and deletions, and small structural rearrangements. It is also used for identification and/or validation of mutation sites (e.g. CRISPR screens).

For such applications, short-read sequencing is often the method of choice, mainly because of the lower costs compared to long-read technologies.

At the DcGC, we have established WGS workflows with or without PCR amplification.

For more information, please check our guidelines.

Targeted Genome Sequencing & Whole Exome Sequencing

Selective amplification of genomic regions followed by high-throughput sequencing allows examining genetic variation in particular loci of interest. Because it doesn’t involve sequencing the entire genome, the costs are substantially lower. At the DcGC, we offer start-to-end workflow for whole-exome sequencing, and can support you with project design and logistics, or any other customized solution.

Whole Exome Sequencing

Targeted approaches can be applied genome-wide, as is the case for whole exome sequencing methods. Relying on sequence-specific capture probes for selective enrichment of exons, it is possible to sequence virtually the entire exome.

We offer whole exome sequencing for both mouse and human samples; please contact us upfront for a discussion on available probe panels most adequate to your experiment.

Targeted Amplification by Multiplex PCR

Targeted sequencing approaches are used when studying mutations in known genomic loci, for example in mutational screening, genome-wide association studies or clinical diagnostics. Genomic loci of interest are enriched with multiplexed PCR and sequenced.

While we do not offer off-the-shelf assays, we can support you in setting up and perform targeted sequencing approaches. Please contact us for further discussions.

16S sequencing

Sequencing of the 16S ribosomal RNA genes allows the identification of biological diversity in samples. This method is often used in metagenomics studies, for example when the aim is to identify microbial composition or identify the diversity of species in a particular environment.

Although we don’t offer library preparation for this type of samples, we have expertise and can advise you on best practices for preparing 16S libraries for sequencing*. We can of course sequence ready-made libraries on short- (Illumina) or long-read (Oxford Nanopore) platforms.

*we offer training in preparation of 16S libraries for ONT sequencing in our OpenLab Unit. Please contact us to discuss this option.

TRANSCRIPTomics

Transcriptomics encompasses a variety of techniques to analyze and quantify gene expression in cells, tissues, or organs. We have significant expertise and offer multiple start-to-end workflows for transcriptome sequencing.

Below you can fine more information about the methods offered by the DcGC for transcriptome sequencing:

Whole Transcriptome Sequencing

Transcriptome sequencing, most commonly known as RNA-seq, captures the entire set of RNA molecules present in a sample at a particular point in time. This is a widely used method to identify, quantify, and compare gene expression in bulk, enabling powerful insights into various biological processes.

At the DcGC, we offer different assays for bulk RNA-seq, and the choice of method depends on the scientific question, the model system, sample quality and material available.

We offer (semi)automated methods for bulk RNA-seq when material is not a limiting factor: one is based on poly-dT enrichment of mRNAs, and the second based on the depletion of ribosomal RNAs. The first method is recommended for samples where the RNA is of high quality and integrity; the second is applied when RNA is of lower quality or when non-polyadenylated RNA species are of interest to your biological question. Our requirements for sample submission differ depending on the method of choice; please check our guidelines for more information.

Additionally, we offer a low-input RNA-seq preparation based on the Smartseq-2 workflow, the method of choice when material availability is limited (e.g. few nanograms).

Please see our guidelines for more information

Immune Receptor Profiling

Sequencing of the immune repertoire (BCR/TCR-seq) enables a better understanding about the composition, function and response of the immune system in various biological contexts. Such information is essential not only for research purposes, but also for diagnostics and monitoring and for development of therapies and interventions.

When combined with expression data from whole transcriptome sequencing, it is possible to contextualize the molecular status of the immune system, giving in-depth insights on the association between the immune system and the overall functioning of cells, tissues and organs.

We offer BCR/TCR sequencing in bulk by selective amplification of BCR/TCR receptors from the pool of cDNA.

Isoform Sequencing

The characterization of the diversity of mRNA isoforms has broad applications in basic and applied research and diagnostics. Different transcript isoforms can be produced by a variety of molecular mechanisms controlling gene transcription, and often specific isoforms can be involved in distinct functions and/or regulatory mechanisms, thus playing important roles in cell and tissue biology in health and disease.

While isoform characterization can be done with short-read sequencing technologies, long-read sequencing platforms such as PacBio or Oxford Nanopore are by far most appropriate, as mRNA molecule are sequenced directly and at its entirety.

At the DcGC, we offer start-to-end workflow for isoform sequencing using the PacBio platform. We can also support projects aiming for the Oxford Nanopore platform in the context of our OpenLab. Please contact us for an in-depth consultation and discussion of your project.

epigenomics

Epigenomics techniques enable to identify and quantify genome-wide the epigenetic landscape of cells, tissues and organs. There are a variety of assays that enable the identification of various types of epigenetic modifications, and this information is ultimately fundamental for a more comprehensive understanding of gene expression. We have significant expertise in many epigenomic assays, but for most of them we only offer partial preparation.

Below you can find more information about the methods offered by the DcGC for epigenomics:

ATAC-seq

Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) is used to map regions of the genome where the chromatin is accessible to binding of transcription factors or other proteins associated with gene regulation – thus, being considered a proxy for regulatory regions.

This method relies on a Tn5 transposase enzyme pre-loaded with sequencing adapters. The enzyme binds to and cuts genomic loci that are accessible for protein binding, integrating the adapters into the cut sites. Interestingly, giving appropriate sequencing depth, this method even allows the identification of transcription factor footprints, since these loci where transcription factors are bound to are not accessible to the Tn5.

We offer a partial service, starting from amplification of the fragmented and tagged DNA. Tagmentation should be done by users.

Please see our guidelines for ATAC-seq.

ChIP-seq

Chromatin immunoprecipitation followed by high-throughput sequencing is a method used to investigate DNA-protein interactions genome-wide.

The assay relies on an antibody-based pull-down of DNA-protein complexes, resulting in the enrichment of genomic fragments that are bound by a protein of interest (e.g. transcription factors or chromatin architecture modifiers) or by proteins with specific chemical modifications (e.g. acetylation/methylation of different histone aminoacids).

We offer a partial service, starting from library preparation. The ChIP pull-down should be done by users.

Cut&Run | Cut&Tag

Cut & Run and Cut & Tag are two antibody-based techniques that make use of enzymatic cleavage of the DNA for identifying protein-DNA interactions. While both assays are designed for targeting specific DNA-bound proteins or chemical modifications of proteins, the enzymes used for DNA fragmentation are different: Cut & Run uses MNases while Cut & Tag uses a hyperactive Tn5 tagmentase enzyme.

Cut & Run is usually more robust in terms of cell types and targeted proteins, while Cut & Tag can be less reliable when the aim is to identify chromatin-associated proteins (e.g. transcription factors) and can be more difficult to optimize. The advantage of Cut & Tag is that it bipasses traditional library preparation as Tn5 is pre-loaded with sequencing adapters.

We offer a partial service for both Cut & Run and Cut & Tag, starting from library preparation.

Methylation sequencing

Modification of cytosine bases by addition of methyl group is an important epigenetic modification that can lead to enhancement or supression of gene expression.

The detection of methylation patterns genome-wide can be done via chemical (bisulfit treatment) or enzymatic (using deaminases) convertion of unmethylated cytosins into uracil. The uracil bases are then read as thymines during sequencing, enabling the identification and quantification of methylated and unmethylated Cs genome-wide.

We offer a start-to-end service for enzymatic methylation sequencing, and a partial service for bisulfit seq, starting from library preparation.

Single-cell sequencing applications

Single-cell sequencing applications are used for profiling individual cells and identifying heterogeneity within cell populations.

TRANSCRIPTOMICS & EPIGENOMICS

The identification and quantification of gene expression at the single-cell level gives valuable insights on cell heterogeneity, allowing, for example, the characterization of the various cell subpopulations and their composition in healthy and disease states, and the identification of novel/rare cell types.

It is possible to combine transcriptome data with other layers of molecular information, such as chromatin accessibility and proteomics, enabling a multi-omics overview of biological systems.

At the DcGC, we have significant expertise in droplet- and plate-based methods for single-cell profiling, and offer multiple start-to-end workflows.

Below you can fine more information about the methods offered by the DcGC for single-cell profiling:

Single-cell RNA-seq

There are various different methods for an unbiased profiling of gene expression at the single-cell level. The choice of the most appropriate – and also most cost-effective – method depends heavily on the biological question, the experimental setup, and the type, quality and number of cells available.

Plate-based assays are generally low-throughput, as cells are sorted into individual tubes or individual wells of 96 or 384-well plates. These are most appropriate for, e.g. initial pilot studies, or when the interest is in specific sub-population of cells that can be enriched and sorted via FACS, or even coupled with laser capture microdissection techniques. In this case, each cell is processed individually and can later be identified by the tube/well-specific barcodes. However, there are also plate-based methods that enable higher throughput (hundred thousands up to one million cells), such as those relying on split-pool barcoding, or combinatorial indexing. The successive rounds of barcoding guarantee that transcripts of each individual cell get a unique barcode combination.

Droplet-based assays generally allow higher throughput, in the range of tens of thousands of cells. Different from plate-based methods, they require live cells* as input, and for this reason require significant optimizations of dissociation protocols. In droplet-based methods, individual cells and barcoded beads are encapsulated together within droplets, which serve as containers for the initial reaction steps where mRNA molecules from individual cells are captured and tagged with a bead-specific barcode. It is possible to increase throughput with a multiplexing strategy, where samples are pre-labelled with barcoded antibodies for specific proteins or with modified membrane lipids and later combined for encapsulation.

*cell fixation is also supported, see more information here.

Needless to say, the type of sequence (full or partial mRNA) captured by each method is different.

We offer start-to-end workflows for a wide range of single-cell assays.

Please see our various guidelines for single-cell RNA-seq.

sc BCR/TCR-seq

Sequencing of the immune repertoire (BCR/TCR-seq) at the single-cell level is possible both with plate- or droplet-based methods, and is often accompanied by full transcriptome sequencing.

Cell surface protein & antigen specificity

Cell surface proteins and antigen specificity can also be analysed and quantified in individual cells by using barcoded antibodies for proteins of interest and barcoded antigens/peptide multimers to match specificity of B/T receptors to such antigens.

We offer start-to-end assays for profiling the adaptive immune system. Labeling steps (for protein or antigen mapping) shoud be performed by the users. Please check our guidelines for relevant information on available antibodies and labelling protocols.

scATAC-seq

The analysis of the epigenomic landscape of individual cells is currently only possible with droplet-based methods at the DcGC.

The method relies on the tagmentation of accessible regions of the chromatin using a tagmentase enzyme, similar to the bulk ATAC-seq method. Thus, results are significantly dependent on the quality of the nuclei in the first place.

The tagmentation itself is done in bulk, in intact and live cells, which are then encapsulated with barcoded beads into gel emulsion droplets containing a single cell-bead pair. The DNA fragments are released with lysis of the cell, and bind to the barcoded oligos attached to the bead. These are then amplified following by addition of sequencing primers and indexes.

As with the droplet-based scRNA-seq assay, it is possible to sequence 10 thousand cells simultaneously. However, sample multiplexing is not yet supported at DcGC.

Please see our guidelines for the droplet-based scATAC-seq.

Multiomics

As part of the droplet-based assay portfolio of the DcGC, we also offer methods to simultaneously quantify different molecular profiles of individual cells, for example the transcriptome and epigenome, or the transcriptome and selected cell surface proteins. Multiomics methods thus enable the integration of different layers of information, giving a more comprehensive view of the cell’s biology and physiology.

The most used assay at the DcGC is the scMultiome profiling, a droplet-based method that combines transcriptome and epigenome profiling. Please read our guidelines on this method.

In addition, we are open to more experimental approaches, being happy to discuss potential collaborations for full or partial support of other multiomics methods.

Spatial omics

Spatial applications provide molecular information of individual cells while preserving the tissue context, enabling understanding of cell heterogeneity in space.

TRANSCRIPTOMICS & PROTEOMICS

Spatial omics is the study of how genes are expressed in tissue space, providing insights into the spatial organization of cells and cell states across the tissue. It introduces a paradigm shift in the way we study gene expression.

While single-cell sequencing can give in-depth information on cell and tissue physiological and metabolic states, it is known that a same cell type can exhibit different transcriptional outputs depending on tissue context. Thus, understanding the link between spatial organization and gene expression programs is especially relevant for a precise understanding of cellular heterogeneity and its microenvironment.

Such type of analysis is done with methods that allow the identification of transcripts and proteins while maintaining tissue context.

At the DcGC, we offer different options for spatial profiling of cells in tissues, so far all using the 10X Genomics platform. The options differ with respect to readout (sequencing or imaging-based) and resolution (multi-cell or subcellular).

In addition, we developed a highly structured collaboration with the Histology and Imaging Technology Platforms of the Center for Molecular and Cellular Bioengineering (CMCB) to provide streamlined workflows from sample preparation to data analysis.

Below you can find more information about the methods offered by the DcGC for spatial omics:

Sequencing-based readout

The technology for sequencing-based readout is marketed by 10x Genomics as Visium, and relies on poly-dT or probe-based capture of the mRNAs. Both enable capture of the full transcriptome in a species-agnostic (poly-dT capture) or species-specific (probe-based capture) manner.

Both approaches make use of spatially indexed slides containing barcoded primers distributed in delimited spots. The standard Visium slides have 55µm diameter spots, thus representing a resolution of 10-20 cells. Visium HD has 2µm spots, enabling subcellular resolution. The species-agnostic version that makes use of poly-dT mRNA capture is only available for the Visium standard, thus not single-cell resolution.

Tissue sections are placed on the slides and permeabilized so that the mRNA (or the probes, in case of the probe-based approach) is captured by the poly-dT primers. After reverse transcription, the resulting cDNA contains the spatial barcode that allows to track back the original location of each mRNA molecule.

The Visium workflow can be applied to fresh frozen, fixed frozen, FFPE and archived sections. In the case of human FFPE samples, it is possible to simultaneously detect immune proteins with a Human Immune Cell Profiling Panel composed of 35 antibodies. Custom probe detection is possible for both mouse and human samples.

Please contact us for support in planning your experiments.

Imaging-based readout

The technology that provides an imaging-based readout is marketed by 10x Genomics as Xenium, which is a sophisticated high-throughput in-situ hybridization that enables the targeted mapping of hundreds of RNA molecules, including isoforms and gene fusions, at subcellular resolution.

This system is based on barcoded, gene-specific padlock probes that hybridize to the RNAs of interest. These barcoded probes are amplified by a rolling circle amplification mechanism, allowing the gene-specific barcodes to be detected by imaging over successive rounds of hybridization with fluorescent-labeled probes.

The Xenium workflow relies on tissue-specific gene panels, thus not allowing the quantification of the entire transcriptome. Different versions of the workflow are available, allowing for capture of 480-5.100 genes simultaneously with custom or off-the-shelf panels.

The assay can be applied to fresh frozen and FFPE samples.

Please contact us for support in planning your experiments.

Sequencing applications

Our sequencing-only services are a cost-efficient manner of sequencing libraries prepared elsewhere.

Sequencing-only services

With our large-scale instruments, we can offer cost-efficient platforms for sequencing samples which need to be prepared with workflows not offered by our lab, or when researchers need some level of flexibility during preparation due to sampling or other aspects related with the experimental model or experiment design.

Most types of libraries are usually compatible with our own preparations and sequencing systems, however, depending on the type of library, or particularities of its design, it might be necessary to allocate dedicated sequencing capacities.

As a flawless library preparation is critical for the success of the sequencing run, and hence for obtaining good sequencing data, we strongly advise to contact us before starting your experiments. This way we can help with chemistry components and a proper indexing strategies, which are crucial for planning the sequencing itself.

Please also take the time to read our guidelines, and do get in touch to discuss your needs.