Genomics
The Genomics Unit aims to provide services in different experimental methodologies for the study of DNA and RNA to researchers from the University of Vigo and other public and private centers and institutions. The Genomics Unit offers researchers different techniques for studying the genome. On the one hand, the Unit offers DNA and RNA sequencing, both by the Sanger method with subsequent capillary electrophoresis in the genetic analyzer and massive sequencing of the same (NGS) using semiconductor technology on Ion Torrent chips. For these methodologies, the entire experimental process is carried out in this unit (sequencing reaction and subsequent purification and analysis, as well as the preparation of NGS libraries and their sequencing).
On the other hand, the unit has equipment for conventional real-time PCR and digital PCR (dPCR). With this equipment, researchers can make absolute and relative quantifications (gene expression) of DNA and RNA and detect gene variants in very low proportions (rare alleles). For both internal processes and external users, this unit also has equipment intended for controlling the quantity and quality of genetic material. In addition to a spectrophotometer and fluorimeter, there is a bioanalyzer that allows, through capillary electrophoresis on a chip, the calculation of RNA integrity (RIN) and the quantification of libraries for NGS.
In the Genomics Unit, in addition to the experimental work described above, the technical staff also provides advice to researchers and users on the application of these instrumental techniques to their projects and lines of research.
Instrumental Facilities
SEQUENCIATOR CAPILAR SEQSTUDIO GENETIC ANALYZER
This equipment has 4 capillaries and can be used for the following applications: de novo sequencing and resequencing (mutation profiling), microsatellite analysis, AFLP, LOH, SNP detection and “SNP screening”.
NEXT GENERATION SEQUENCERS (NGS)
The Ion torrent sequencer available is the Ion GeneStudio S5 Plus System. With this equipment, sequencing with data outputs from 0.3-0.5Gb to 40-50 Gb can be carried out, simply using different chips. In addition, we also have the Illumina MiniSeq System sequencer which has a data output of 1.65-7.5Gb.
ION CHEF
This equipment is a robot for template preparation and chip loading for massive sequencing. Additionally, it allows the preparation of AmpliSeq libraries
THERMAL CYCLER 7900 HT FAST REAL-TIME PCR
This equipment allows PCR amplification of DNA fragments and their real-time monitoring using fluorescent probes (TaqMan probes) or intercalating fluorochromes (SYBR GREEN). In addition to the 96-well plate block, we also have a block for low-density arrays (TLDA’s).
THERMAL CYCLER 7900 HT AND QUANTSUDIO 6 PRO REAL-TIME PCR
These equipments allow PCR amplification of DNA fragments and their real-time monitoring using fluorescent probes (TaqMan probes) or intercalating fluorochromes (SYBR GREEN). In addition, the 7900HT thermal cycler has a block for low density arrays (TLDA’s).
QUANTSTUDIO 3D DIGITAL PCR SYSTEM
PCR is performed on a chip containing 20,000 piglets (Digital PCR Chip v2), where the sample and fluorescent reagents (TaqMan® or SYBR GREEN™ probes) are loaded onto the ProFlex™ 2x Flat PCR System. The fluorescence reading from the chip takes place on the QuantStudio 3D and the data analysis is done in the cloud.
AGILENT TECHNOLOGIES BIOANALYZER 2100
This equipment allows to quantify, integrate and check the quality of DNA, RNA and protein preparations. In addition to the quantity values, it also offers the RIN value for RNA samples.
NANODROP ND-1000
is a spectrophotometer (range 220 to 750 nm) that allows quantification and calculation of DNA and RNA purity values in sample volumes of 1 to 2 microliters.
QUBIT 4 FLUORIMETER
This equipment allows highly specific quantification of DNA and RNA samples in a volume range between 1 and 10 microliters using fluorescent probes.
Applications
- Sequencing of gene regions (amplicons, plasmids)
- DNA fragment analysis (microsatellites, STRs, VNTRs, etc)
- Analysis of point mutations and polymorphisms
- New sequencing (small genomes) and resequencing
- Sequencing of exomes, transcriptomes and small RNA
- Metagenomics
- Sequencing of target gene panels or on demand (AmpliSeq DNA/RNA)
- Sequencing with designed panels of circulating DNA (cfDNA, OncoMine panels)
- Gene expression and quantification
- Allelic discrimination by TaqMan probes
- Allelic discrimination by high resolution dissociation (HRM)
- Rare allele detection and pattern-free quantification (dPCR)
- Spectrophotometric and fluorometric quantification of nucleic acids
- Quality and quantity control of DNA and RNA (RIN calculation)
Sample requirements
Users must submit a document with the number and name of the samples to be analyzed in the request. In addition, this document will indicate the nature of the samples (DNA or RNA) and all the information available: size of the amplicons, size of the inserts in plasmids, concentration of the sample if known, solvent in which they are dissolved, purified with ExoSap or not, etc. Each sample must be perfectly identified with its name (which should preferably be a number).
The volume and concentration of the sample will depend on the application to be performed. For quantification, a minimum volume of 5 µL is necessary (including the Bioanalyzer). For Sanger sequencing, between 100 and 300 ng is necessary, so the volume of the sample will be a function of its concentration. The recommended amount would be between 10 and 15 µL of sample at a concentration of 40-50 ng/µL. Samples for migration in the capillary sequencer must come already prepared resuspended in formamide with incorporated size marker (for fragments) or purified and resuspended in formamide or water for sequences.
Samples for mass sequencing will first be evaluated in quantity and quality. The volume and amount of DNA for NGS will depend on the application, so it will be established at each moment by the staff of this unit, after consultation with the users. As an indicative rule, the minimum recommended amount of DNA for NGS will be 40-60 ng/µL (with a minimum volume of 20 µL), although they may be lower depending on the application. The DNA can be resuspended in TE, Low TE, EB, etc., although its resuspension in water is recommended. Regarding the quantity and quality of RNA for NGS, the same will happen, although samples with RIN values lower than 6.5-7 will not be sequenced unless previously agreed with the users. The preparation of standard libraries for transcriptomics should start from a minimum of 100 ng, although it is recommended to have a minimum of 500 ng. The RNA should preferably be resuspended in water.
Any questions users may have about the quality or quantity of their samples will be discussed with the unit’s staff.