Genomics and Bioinformatics Unit

Next Generation Sequencing (NGS) has been a revolution in the field of genomics, thanks to its high throughput compared to more traditional technologies, offering a wide variety of applications to study biological systems at a more complex level.

The platforms developed by Illumina are based on sequencing by synthesis (SBS) in which the detection of labelled nucleotides is performed as they are incorporated into the chain being formed. Specifically, the two systems we have in the Genomics Unit allow us to sequence fragments from 36 bpb to 600 bpb, and a sequencing depth ranging from 1 to 400 million reads per run. This results in accurate data for a wide range of applications:

Genomics (DNA-Seq):

• Sequencing of small complete genomes (microorganisms, viruses).
• Sequencing of exomes and large panels (enrichment-based).
• Targeted gene sequencing (gene panels, amplicons).
• De novo sequencing.

Transcriptomics (RNA-Seq):

• Gene expression profiling (Total RNA-Seq, mRNA-Seq).
• Targeted sequencing of expression profiling.
• Analysis of small RNAs (miRNA and Small RNA).

Epigenomics:

• Methylation analysis (MeDIP-Seq, RRBS-Seq).
• DNA-protein interaction analysis (ChIP-Seq, HI-C/3C-Seq, 4C-Seq).
• RNA-protein interaction analysis (RIP-Seq, CLIP-Seq).
• Retrotransposon capture analysis (RC-Seq).

Metagenomics:

• Metagenomic profiling (microbiome studies).
• Sequencing of 16S, 18S, ITS subunits (ID of microorganisms).

The basic NGS project development service will include:

• Initial quality control of extracted samples.
• Standardisation.
• Library preparation.
• Library validation by high resolution electrophoresis.
• Pooling or multiplexing.
• Sequencing in NGS equipment.
• Demultiplexing and Raw Data delivery (FASTq).

Genotyping is used to determine the genetic information of an organism, genotype, highlighting the differences between different genomes. Depending on the type of study to be carried out, the Genomics Unit has different types of technologies to cover from small and medium scale studies to studies that require high-throughput technologies that allow genome-wide association analysis (GWAS).

Microarray technology provides a valuable tool for the detection of genetic variants. In addition, the format in which they are presented allows the processing of large sample volumes in a short time. Illumina® has a wide variety of pre-designed kits for the genotyping of human and non-human DNA, as well as for the analysis of DNA methylation patterns, offering also the possibility, in many of them, to customise the assay.

The Genomics Unit covers the possible needs of all researchers with Illumina® pre-designed kits, among which we could highlight:

Infinium Global Screening Array-24 V3.0 Kit

This array has approximately 654,000 markers (with the possibility of adding up to 50,000 more), providing a tool for population-scale genotyping, variant detection, pharmacogenetic studies and precision medicine research.

Infinium MethylationEPIC Kit

The Infinium MethylationEPIC array can quantitatively interrogate more than 935,000 methylation positions in the human genome at single nucleotide resolution. It allows analysis from FFPE paraffin-embedded samples.

Infinium CytoSNP-850K v1.2 BeadChip Kit

This kit encompasses a list of genes based on the content of the International Collaboration for Clinical Genomics (ICCG) and the Cancer Genomics Consortium (CGC), so that, with up to 850,000 markers, it provides a comprehensive overview of genes of recognised cytogenetic relevance in both conventional and cancer applications.

For more information on the possibilities offered by the company, please visit https://www.illumina.com/index-d.html.

The Illumina® microarray genotyping and methylation analysis service includes:

• Quality control of extracted samples: verification of quality and concentration.
  Standardisation.
• Amplification of genomic DNA, enzymatic treatments, array marking, scanning in the equipment and data collection.
• Generation and sending of preliminary analysis results.

Digital PCR is a new approach to DNA detection that provides absolute quantification and target detection more efficiently and accurately than quantitative PCR, all without the need for reference standards or standards. Since its appearance, it has been essential for detecting rare variants, fusion proteins and for liquid biopsy work.
In the Genomics Unit we have the Applied Biosystems™ QuantStudio Absolute Q Digital PCR System to carry out digital PCR reactions, which generates absolute quantification data with high precision and sensitivity.
Applications of this technology include:

• Detection and quantification of CNVs with high precision.
• Detection and quantification of rare alleles.
• Absolute quantification of bacterial and viral load, and absolute pathogen counts.
• Absolute quantification of NGS libraries and validation of sequencing results, without the need for reference standards.
• Detection of gene expression changes for absolute quantification of transcription.
• Detection and absolute quantification of mutations in plants and genetically modified organisms.

Real-time PCR is a widely used technique for numerous qualitative and quantitative applications. We have up to 4 different platforms to perform these assays: ABI Prism 7900HT, ABI Prism 7500, QuantStudio™ 6 Flex Real-Time PCR System and QuantStudio™ 12K Flex System, from Applied Biosystems™.

The services offered in this category would include:

• Design of optimal primers and probes, depending on the nature of the assay to be performed.
• Selection of candidate genes in the different databases, according to a particular category such as biological function, cellular location, chromosomal position, etc., of interest to the user.
• Identification of single nucleotide polymorphism (SNP) genotypes in allelic discrimination studies.
• Analysis of copy number variation (CNV) of a given gene.
• Determination of the expression levels of transcripts of interest (mRNA) by RT-qPCR.
• Splicing variations: selection of annotated exons, primer design, RT-PCR and electrophoresis.
• Analysis of microRNAs, using specific primers.
  High Resolution Melting (HRM) analysis, either to detect differences in methylation between genes (MS-HRM) or to search for mutations in target sequences.

Also based on real-time PCR, OpenArray™ technology allows you to perform gene expression analysis, SNP genotype identification, microRNA analysis or pharmacogenomics assays through pre-designed or new TaqMan™ assays that can be selected to suit your experiment requirements.

Depending on the analysis to be performed, there are up to 11 pre-designed experiment set-up possibilities adapted to our Applied Biosystems™ QuantStudio™ 12K Flex System.

For more information on experiment design, please visit the ThermoFisher website: https://www.thermofisher.com/es/es/home/life-science/pcr/real-time-pcr/real-time-openarray/open-array-panel.html.

With Agilent’s Bioanalyzer 2100 and TapeStation 4200, the quality of nucleic acids, both DNA and RNA, can be determined automatically by means of a high-resolution electrophoresis process.

This procedure has become an essential part of many genomics laboratory workflows, especially as quality control of the quantity, integrity and purity of DNA and RNA in NGS library generation processes, requiring very small sample volumes.

Applications of nucleic acid analysis on Bioanalyzer 2100 and TapeStation 4200:

• Quality control of DNA/RNA libraries throughout the NGS workflow: quantification and fragment size.
• Quality control and integrity assessment of RNA samples.
• Analysis of microRNA content in TotalRNA samples.
• DNA quality control in FFPE samples.
• Optimisation of qPCR assay design.

Sonication

Sonication equipment has become an essential instrument in any laboratory dedicated to genomics research. With the E220 Focused-Ultrasonicator, from Covaris®, we achieve high precision in the control of the whole process thanks to the AFA technology that manages to direct the wave to the centre of the well.

Among the applications that this technology allows us are:

• Fragmentation of DNA/RNA molecules, with adaptation of the cycles and times according to the desired molecule size.
• Chromatin fragmentation.
• Extraction of nucleic acids and proteins.
• Homogenisation of samples.

End-point PCR

In the Genomics Unit we have several pieces of equipment for conventional PCR: Veriti™ Dx Thermal Cycler and GeneAmp® PCR 9700, from Applied Biosystem™.
This equipment is distributed in two physically separate areas to cover the full range of applications in which they are needed. Thus, a pre-amplification area (free of amplicons), where they will be used in processes such as NGS library generation, incubation periods in sample purification protocols, bisulphite conversion process for methylation pattern studies, etc. Those located in the post-amplification area will be used for the amplification of regions of interest of the genome under study.

Spectrophotometric and fluorometric analysis of nucleic acids

Almost all the applications that are developed in a genomics laboratory require previous guidelines that give information about the quality and concentration of the samples.
We have different equipment to obtain this information:

• NanoDrop™ 2000c (ThermoFisher Scientific), UV-visible spectrophotometer, which from a minimum sample volume (0.5-2 µL) gives us information on the concentration of DNA, RNA, proteins, etc., in addition to evaluating the purity and quality of the solutions through the 260/280 and 260/230 ratios.
• Qubit 4 (ThermoFisher Scientific), fluorometer that uses fluorescent probes for binding to DNA, RNA, proteins, etc., providing a more specific measure of sample concentration.

NextSeq 500 (Illumina®), is a new generation platform for massive DNA sequencing. It allows the sequencing of whole genomes, exomes, targeted panels and transcriptomes, among others, with very high throughput, based on the recognized “Sequencing By Synthesis” (SBS) chemistry.

The NextSeq 500 system is very versatile. It allows medium- to large-scale sequencing, making it adaptable to a multitude of different projects without compromising its optimal throughput and efficiency. It enables gene expression profiling studies, mRNA-Seq, small RNA analysis, epigenomics studies, targeted gene sequencing, exome and large panel sequencing, as well as whole genome sequencing.

MiSeq (Illumina®), is another NGS platform also based on SBS technology, capable of delivering high-precision data and robust performance for a wide range of applications: targeted gene sequencing, small genomes and amplicons, 16S metagenomics, etc.

It is capable of paired-end reads up to 15 Gb per run, providing sequence data of more than 600 bases per read. A compact all-in-one platform that also incorporates data analysis and access to Sequence Hub de BaseSpace™ (Illumina’s genomic analysis platform).

The iScan platform (Illumina®) is a high-resolution microarray scanner based on Bead-Based microarray technology.

It features microarrays covered by hundreds of thousands of microbeads carrying copies of specific oligos. These act as capture sequences for a wide range of DNA analysis applications.

The iScan system is accompanied by a wide variety of assays and kits for whole genome or targeted genotyping, methylation profiling and cytogenetic analysis, available from the Illumina website: https://emea.illumina.com/systems/array-scanners/iscan/products-services.html.

Complementing the workflow of this technology is the Infinium Automated Pipetting System with ILASS (Infinium Automated Pipetting System with ILASS). This advanced device represents the ultimate in automation for low to high volume genotyping and methylation analysis projects.

It is a high-precision instrument designed to automate specific steps within Illumina’s Infinium array workflows. The system is fully compatible with Infinium EX BeadChips (Infinium Global Screening Array-48 v4.0, Infinium Global Clinical Research Array-24 v1.0, Infinium Methylation Screening Array-48), the brand’s most powerful array format, enabling seamless integration with the Unit’s existing protocols.

Two QuantStudio™ 6 Flex Real-Time PCR Systems, Applied Biosystems™ with 96- and 384-well blocks. The QuantStudio systems feature OptiFlex technology, which allows advanced multiplexing for quantification of multiple genes within the same experiment. This system allows both TaqMan and SYBR-based assays to be performed in the same sequence, facilitating greater flexibility in experimental design.

QuantStudio™ 12K Flex System with OpenArray™. Enables analysis of 1 to 12,000 reactions in a single run, and up to 110,000 reactions in a single day, when combined with the AccuFill QuantStudio 12K Flex OpenArray System. It’s a technology that lowers cost and reduces handling time.

Two Applied Biosystems™ 7900HT platforms, with 96- and 384-well blocks and a microfluidic card block. Real-time PCR systems capable of 1-30 position analysis on 384 samples. In addition, an Applied Biosystems™ 7500 real-time PCR system, platform calibrated for a wide range of available fluorophores: FAM™⁄SYBR™ Green I, VIC™⁄JOE™, TAMRA™⁄CY3, ROX™⁄Texas Red and CY5.

Applied Biosystems™ launches its new digital PCR system, the QuantStudio Absolute Q Digital PCR System, in 2022. This new platform is based on microfluidic array plate (MAP) technology that allows to concentrate most of the workflow on a single instrument (compartmentalization, thermal cycling and data acquisition), minimizing hands-on steps and maximizing assay uniformity.

• Thermal Cyclers:
  – Six VeritiTM Dx Thermal CyclerTM systems.
  – SimpliAmpTM Thermal Cycler System
  – Two GeneAmp® PCR 9700 systems
• DNA, RNA and protein quantification systems:
  – Bioanalyzer 2100 (Agilent Technologies).
  – 4200 TapeStation (Agilent Technologies)
  – NanoDrop™ 2000c (Thermofisher Scientific)
  – Qubit™ 4 (Thermofisher Scientific)
  – Two ImageQuantTM LAS 4000 Systems (GE Healthcare Life Sciences)
• Robot:
  – Freedom EVO.100 Robot (Tecan)
  – Three QIAcube Connect Automated Nucleic Acid Extraction Systems (QIAgen)
  – Maxwell Automated Nucleic Acid Extraction System (Promega)
  – E220 Focused-wave Ultrasonic Focused-ultrasonicator (Covaris)
  – Automated liquid distribution for sample preparation
• Hoods and safety:
  – AURA laminar flow hood, with EPA filter and UV light.
  – ASECOS safety air extractor cabinet.
  – Extraction hood type 1, SCALA.