Translational genomics

MiSeq® Personal Sequencer (Illumina): Mass sequencing device that can generate up to 20 Gb per run, 20 million reads from 2 x 300 bp in a maximum of 384 indexed samples. It uses Synthesis Sequencing (SBS) technology and consists of amplifying, sequencing, and analyzing data in an instrument using RTA software v1.18.54.0 (Real-Time Analysis), MCS v2.6.1.1 (MiSeq Control Software), MSR. 2.6.2.3 (MiSeq Reporter) and cloud data storage (BaseSpace). ABI Prism 3130 / 3130xl Genetic Analyzers (Applied Biosystems): Sequencers with 4-16 capillaries per 96 w or 383 w plate, CCD detection camera and multiline argon ion laser with primary excitation at 488 and 514.5 nm. Software Acquisition, Data Collection v3.1 and Sequencing Analysis v5.4, GeneMapper v4.1 and SeqScape 2.7. 2200 TapeStation® (Agilent): Automatic electrophoresis system that analyzes nucleic acids (fragments or libraries of DNA, DNA or RNA) or proteins in a capillary support (ScreenTape) and scans images. It allows the calculation of nucleic acid size (in bp) and proteins (kDa), quantification and quality analysis using GeneTools software with a built-in simplified band comparison system. The systems use a very small sample size (1-2 µL) and readings can be made on individual tubes or 96 w plates. qPCR LightCycler®480 in real time (Roche Diagnostics): For 96w and 384w interchangeable board formats. Xenon lamp, emissions 430-630 nm, filters with excitation at 440, 465, 498, 533 and 618 nm and emission at 488, 510, 580, 610, 640 and 660 nm that allow the detection of the most common fluorochromes (FAM, NED). , ROX, SYBR, TAMRA and VIC). LightCycler® 480 program for acquisition and analysis and LightCycler® Probe Design Software 2.0. 1. NextSeqTM-1000: Mass sequencing equipment that can generate up to 120 Gb per use and 400 million reads in 2 x 150 cycle runs. The system uses Illumina synthesis sequencing by synthesis technology (SBS) and can analyze the results locally, by remote connection and with the DRAGEN bio-IT FPGA secondary analysis system MINion System (Oxford Nanopore): Mass sequencing device based on the use of nanopores suspended over a membrane that allows the passage of nucleic acids (DNA and RNA). The bases are identified by calculating the changes in electric current produced in the membrane. Very long readings (> 4 Mb) and up to 50 Gb of data are generated in 72-hour runs at 420 bases / second. Suitable for whole genomes / exomes / metagenomics, directed sequencing, whole transcriptome (cDNA), small transcriptome (direct DNA) and methylation studies. Advanced QIAxcel system This is an automated capillary electrophoresis system that separates DNA and RNA fragments based on molecular weight (up to 96 samples per roll). The analysis software (BioCalculator ™ software) can generate both electropherograms and gel images and can determine: size and concentration of DNA fragments (15 bp to 15 Kb); b) proportion, concentration and quality of total RNA; c) cRNA / cDNA and fragmented RNA / DNA quality. Sample preparation is easy, as the capillaries contain the ethydium bromide needed to visualize the fragments. The amplification is done in a conventional thermocycler that supports a 96 w plate using EvaGreen chemistry or based on hydrolysis probes. The amplification results are read by a QX200 drop reader. It can analyze up to 96 samples and has an optical fluorescence detection system (multipixel photon) in the FAM (EvaGreen) and HEX (alternatively VIC) channels. The device has a linear discrimination range of up to 5 orders of magnitude and an accuracy of ± 10%. QX200 ™ Droplet Digital ™ PCR System: The QX200 Droplet Digital PCR (ddPCR) system is a combination of three devices: The QX200 drop generator divides samples ((20 µl) into 200 drops using microfluidics and an oil emulsion. up to a maximum of 8 samples in 5 minutes. EpMotion 5070 robotic station: A robotic liquid dispensing system with two one- and eight-channel tools that dispense volumes between 1 and 50µl. You can pipette 1-2µl volumes with or without contact. It has an accuracy of less than 2% hp at 1µL. It has an optical sensor to automatically detect and calculate the volume of liquid, laboratory material and tips. It is compatible with tubes (0.2 ml to 50 ml) and microplates of up to 384 wells.

Genetic Analysis Service The genomics platform allows the study of the genetics of organisms through the use of various techniques: Deep sequencing (NGS) This consists of the preparation of libraries and the subsequent high yield of deep sequencing of the region of interest. The possibility of making multiple readings means that rare clonal types, cells or microbes are detected that represent only 1% of the original sample. The libraries are developed using different approaches, all aimed at obtaining fragments of the right size to which barcodes (identifiers in each sample) and the adapters to read them have been attached. Applications: This technology is applicable to: Genomic DNA (which genome, exome) Enriched DNA fragments Simple whole genomes Metagenomic profiles (16 years or others) RNA (transcriptomic, RNA profile) Small RNAs / miRNAs Immunoprecipitated chromatin (Chip-Seq.) Etc. Sequencing is performed on a personal MiSeq® sequencer (Illumina), a NextSeq-1000 (Illumina), and a MinION sequencer (Oxford Nanopore). Capillary electrophoresis for sequencing: These are the gold standard for genetic diagnosis when specific regions of the genome are to be analyzed. They allow both Sanger sequencing and gene fragment analysis (genescan). The most common applications are: de novo DNA sequencing; mutation detection; allele discrimination studies (SNPS); analysis of gene methylation profiles in DNA CpG islands (MSP); analysis of the variable number of gene copies (CNV); microsatellite analysis (STRs, VNTR); Binding PCR and multiplex ligand-dependent probe amplification (MPLA). This is done using ABI 3130 and ABI 3130xl genetic tests (Applied Biosystems). Capillary electrophoresis for the analysis of the quality of nucleic acids To study the quality of nucleic acids we have a 2200 TapeStation bioanalyzer (Agilent) and a QIAxcel Advanced System (Izasa). Both are automated systems for quantitative and qualitative analysis of nucleic acids based on capillary electrophoresis. The applications offered are: g / DNA quantity / quality analysis (including DNA integrity number (DIN), DNA fragments (150-5,000 bp) and RNA analysis in standard format or high sensitivity (including RNA integrity number (RIN)). Amplification of nucleic acids Real-time PCR, based on tracking the fluorescence emitted during the amplification reaction in each cycle simultaneously in all samples. It allows the determination of amounts of DNA, RNA or MiRNAs present in the original sample (qPCR) or the qualitative identification of specific DNA (genotyping). The main applications are: absolute / relative quantification of differential gene expression profiles; allele discrimination studies (SNPS); analysis of gene methylation profiles in DNA CpG islands (MSP); analysis of variable number of gene copies in genomic or viral DNA (CNVs), quantification of gene dose (QF-PCR to detect aneuploidies); High Resolution Melting (HRM) for gene scanning. Digital Droplet PCR (ddPCR) is a technology that fragments each sample into more than 20,000 drops individually wrapped in a layer of oil, within which the polymerase reaction takes place. It can be performed alternating with labeled double-stranded or fluorescent probes and provides an absolute and ultrasensitive quantification of nucleic acids. It is especially useful to quantify low-abundance gene fragments, allele variants (SNPs), viruses, and bacteria underrepresented in the heterogeneous mixture. Etc. Complementary Services Fluorometric quantification with a Quantus ™ Fluometer (Promega). A two-channel fluorometer optimized to measure nucleic acids (RNA and DNA) before and after the generation of sequencing libraries Robotic liquid dispensing using an EpMotion 5070 station (Eppendorf). This can prepare serial dilutions, PCR assays, transfer tube samples to plates (96 w and 384 w), plate reformatting, and so on. There are also consulting services on experimental design, sample preparation and analysis of results and global interpretation of data.

GINYS-IGTP-001

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