Genomics

All in Oeiras

• G400 (MGI) – DNBSEQ-G400 can flexibly support a variety of different sequencing modes, supporting a range of read lengths from SE50, to PE300 and outputs of 55 Gb~1440 Gb per run.
• NextSeq 2000 (Illumina) – It is a very flexible sequencer offering 4 different capacities (130, 400, 1200 and 1800 million reads) with read sizes from SE50 to PE300 (300+300).
• Miseq  (Illumina) –  The ideal sequencer for sequencing amplicons (16/18S and ITS) and for small pilots or when longer reads of 600 cycles (300+300) are required.
• Fragment analyser (Agilent) – Is an automated capillary electrophoresis system from Advanced Analytical (now Agilent). It is capable of measuring, quantifying and assessing the quality of nucleic acids. It can be used for DNA (genomic DNA, NGS libraries) or for RNA. For a small number of samples or for faster analysis, please refer to the TapeStation system.
• Agilent’s TapeStation 4200 (Agilent) – TapeStation is an easy-to-use fluorescence-based electrophoresis instrument for size measurement, quantification and quality control of nucleic acids. It can be used for different types of DNA (genomic DNA, NGS libraries…) or for RNA. For 10 or more samples, see our fragment analyser.
• Chromium X (10 Genomics) – The flexibility of the Chromium X makes it possible to perform low, medium and high throughput (HT) single cell assays or immune profiling and fixed RNA samples (human and mouse only). The instrument’s chip now allows up to 16 samples to be loaded per run, doubling the sample processing capacity. Chromium X offers users more flexibility in assays. By using HT reagents/chips, it is possible to encapsulate up to 20,000 cells or nuclei per individual reaction or up to 60,000 cells/nuclei when using multiplex reagents (Cellplex).
• Biomek i7 (Beckman) – The Biomek i7 Automated Workstation has been designed to optimize dependability and walk-away time in mid- to high-throughput labs. The Biomek i7 Hybrid with enclosure has up to 45 deck positions and includes both Multichannel and Span-8 pipette heads in an enclosed work space.
• PromethION (Nanopore P2S) – A flexible, high-yield sequencing, in a compact, accessible design. Utilising flow cells that generate hundreds of gigabases, enable PromethION-scale benefits in small to medium sized labs. The two flow cell running in pararell can deliver up to 580 Gb of data and up to 2Mb per read.

 

RNA-Seq (Transcriptomics)

The unit offers RNA-seq library preparation, with multiple options such as: ribo-depletion (Bacterial and Eukaryotic), Poly-A enrichment, 3′-Tag-Seq (QuantSeq) libraries and microRNAs. Libraries can be constructed to retrieve the full transcript. 

Sequencing is performed Single End (SE)100bp, PE100 (100+100) and PE150 (150+150). Optional SE50 is available under consulting.

A SMART-Seq2 protocols allow us to produce RNA-Seq libraries from very limited amounts of starting material (up to single cells). Reads can cover complete transcripts and can be used to detect alternative splicing, in addition to regular differential expression.

 

Key features:

• Total RNA input as low as single cells (e.g. from FACS).

• Low-cost library construction with stranded RNA as option
• Flexible output (from 100 Mreads up to 2000 Mreads per flow cell) and scalable pricing.

Requirements:

• 5 to 500 ng of high quality total RNA or 1-300 single cells in RLT+ buffer (inquire for details).

We require total RNA samples. We do not perform RNA isolations in the lab.

Whole Genomics Sequencing (WGS)

The unit has implemented 2 protocols, a tagmentation based for low input samples (~1ng) and a PCR free (~1000ng) .

 

Key features:

• Low input, low cost.

• Can also be used to sequence plasmids or amplicons (e.g. mix of PCR amplified genomic regions of interest).

• Sequencing on NextSeq2000 or MGI G400 (from 2x150bp to 2×300 PE).

Requirements:

• 1 ng of high quality genomic DNA or amplicon(s) larger than 400bp or 1000ng for PCR free libraries.

 

Nanopore Sequencing

Nanopore sequencing with Oxford Nanopore Technologies (ONT) systems enables high-throughput long-read sequencing of both DNA and RNA samples. We offer long reads lib preparation and sequencing on a MinION instrument (PromethION P2 soon), with either MinION or Flongle flow cells.

 Key features:

• Long-read analysis.

• Real-time analysis for rapid, efficient workflows.

• Adaptable to direct DNA or RNA sequencing as well amplicon sequencing (e.g.16S)

Requirements:

• High quality, pure genomic DNA (gDNA).

• Purity as measured using Nanodrop – OD 260/280 ~ 1.8 and OD 260/230 ~ 2.0–2.2.

• Input mass, as measured by Qubit – from 10ng to 1000ng. Output yield dependent of protocol and starting material.

 

Spatial Transcriptomics

The unit offers 10X Genomics Visium Spatial Transcriptomics service and STOmics Stereo-Seq with the Histopathology Facility. This allows for transcriptome-wide mapping of genes expression onto tissue sections, with concomitant H&E or IF staining. The option to process both FFPE slides (human and mouse only) or Fresh frozen tissues is available. Please contact us for more details.

Amplicon sequencing (16S, ITS and 18S)

Classical 16S V4 rRNA sequencing

The 16S metagenomic protocol focuses on the V4 region of bacteria 16S region (primers 515F-806R) and is designed to amplify from bacteria and archaea (Walters et al., 2016). 

The ITS protocol detailed here is designed to amplify fungal microbial eukaryotic lineages using paired-end community sequencing on the Illumina platform with primers ITS1f-ITS2 (EMP.ITSkabir).

The 18S protocol detailed here is designed to amplify eukaryotes broadly with a focus on microbial eukaryotic lineages. The primers target the 18S SSU rRNA and are based on those of Amaral-Zettler et al. (2009) and Stoek et al. (2010). 

Using a 280-multiplex approach on a 2x250bp PE MiSeq run we deliver up to >50.000 reads per sample. Other primers combinations on request (e.g. V4-V5;V1-V2 and V1-V3)

 

Key features:

• 3 independent PCRs per sample to reduce bias.

• High multiplex to reduce cost per sample.

Requirements:

• 10 ul of each sample with a concentration of up to 100 ng/ul.

• If you’re planning to deliver more than 20 samples, please consider to use a PCR plate (96 wells, non-flat bottom) instead of tubes. Sample orientation A1->A12.

• For low biomass samples (< 10ng/ul) please contact us.

Express Service (V3-V4) and or ITS (96 samples)

Contact us for more information.

 

scRNA-SEQ

10x Genomics is a single-cell profiling technology is becoming the most popular alternative for the analysis of large cell numbers. The platform allows for high-throughput analysis in a variety of cell types as well as single-cell nuclei. The workflow encapsulates cells or nuclei together with gel beads into nanodroplets. Different protocols are available: Gene Expression (GEX), scATAC, GEX+immune profiling and multiome (GEX + ATAC). Preliminary Single-cell data analysis is provided by Cell Ranger and Loupe Cell Browser software.

 

Possibilities: 

• Feature Barcoding can be useful to look for certain proteins on the cell surface (CITE-seq) or using markers in order to pool different samples into one channel (Cell Multiplexing/Cell Hashing). Cell plexing can be also achieved by using molecular tags.
• Sequencing of full single cell trancriptome by nanopore sequencing

For any queries please contact us.

 

Nucleic Acid QC

Our Nucleic Acid QC service is based on 2 instruments: Fragment analyser and TapeStation both from Agilent.  Fragment analyser is more suitable for a large number of samples and TapeStation for fast QC acquisition and small number of samples ~7-8. Fragment analyser is more sensitive for low amounts of RNA/DNA while TapeStation can discriminate the sample quality in RIM (RNA) or DIM (DNA).

 

Fragment analyser 

DNA – High Sensitivity NGS
DNA Fragment Concentration Range: 5 pg/uL – 500 pg/uL; DNA Smear Concentration Range: 50 pg/uL – 5000 pg/uL

RNA – High Sensitivity RNA
Quantitative Range (per smear) (Total RNA) 50 pg/uL – 5000 pg/uL; (mRNA) 500 pg/uL – 5000 pg/uL

TapeStation:

DNA – D1000: DNA Sizing Range: 35 bp – 1000 bp
Sample Concentration: 0.1 ng/µL – 50 ng/µL input DNA

DNA – D5000: DNA Sizing Range: 100 bp – 5000 bp
Sample Concentration: 0.1 ng/µL – 50 ng/µL input DNA

DNA – Genomic: Sizing Range: 200 bp – 60000 bp
Sample Concentration: 10 ng/µL – 100 ng/µL input DNA

RNA – High Sensitivity: Sizing Range: 100 bp – 6000 bp
Sample Concentration: 0.5 ng/µL – 10 ng/µL input RNA