Next Generation Sequencing

Next Generation Sequencing at the Laboratory for Genomics and Epigenomics (LAGE)

PromethION 24
PromethION 24 offers direct, real-time DNA and RNA sequencing technology capable of long readings (>4Mb) of single molecules. The PromethION 24 system is designed to run 24 flow cells, allowing for on-demand sequencing, using any flow cell at any time. The running of single experiments can be started and stopped as required or multiple flow cells can be deployed onto single experiments for greater speed or throughput. Each flow cell allows up to 3,000 nanopores to be sequenced simultaneously, with a yield of > 4.8 Tb (up to 7Tb). The PromethION benefits from the inclusion of on-board compute which permits device control, data acquisition, basecalling and data streaming, all without placing any additional burden on existing IT infrastructure.
Softwares
MinKNOW v22.10.7
EPI2ME Labs 3

MiSeqDX
A massive parallel sequencing platform to be used both in certified IVD (In Vitro Diagnostic) and RUO (Research Use Only) analysis. Illumina’s MiSeqDX System connects Illumina’s sequencing by synthesis (SBS) technology with a workflow that enables researchers to obtain analysed data from DNA samples in as little as eight hours (8h), producing up to 15 gigabases of data in a single instrument run, 25 million reads with a length of 2×300 bp each and base call accuracy parameters greater than 99.9%. MiSeq incorporates cluster generation, sequencing and data analysis functions on a single instrument with full automation and flexibility to process limited numbers of samples.
System specifications
Max read length 2 × 300 bp
Output (2 x 150bp run) ≥ 5 Gb
Reads per run 12 – 25 million
Output range 540 Mb – 15 Gb
Softwares
MiSeq Control Software v3.1.0.13
Sequencing Analysis Viewer v2.4.7

iScan System
An innovative array scanner that provides fast, sensitive and accurate imaging of Illumina BeadChips. The iScan system is part of Illumina’s comprehensive portfolio of innovative assays for genotyping, copy number variation (CNV) analysis and DNA methylation. The scanners and components are modular, creating a system that can be synchronised and configured in accordance with varying levels of required sample throughput. The iScan system uses high performance lasers, optics and sensing systems to provide submicron resolution and high throughput. As a result, scan times are extremely fast while preserving data quality and reproducibility.
With a high signal-to-noise ratio, high sensitivity, a low limit of detection, and a broad dynamic range, the iScan System produces exceptional data quality for use in a wide range of biomarker screening or validation studies. The high call rates (> 99% with the Infinium Assay) enable powerful population screening studies and high-resolution CNV analysis, accurately detecting even single copy number changes. The iScan System is ideally suited for fast, accurate screening in agrigenomics or for complex disease validation studies.
Softwares
iScan Control Software v4.0.0.147
GenomeStudio v2.0.5
llumina Decode file client v3.0.2
Sequencing Analysis Viewer v2.4.

NovaSeq 6000
A flexible, ultra-high throughput DNA sequencing system capable of sequencing up to 48 human genomes at a 30-fold depth in a single instrument run. The instrument offers multiple flow cell formats, various read length configurations and the ability to analyse one or two flow cells at the same time to obtain data output ranging from approximately 160 Gb to 6 Tb and up to 20 billion reads per run, providing flexibility across a wide range of study applications and sizes.
System specifications
Output range 80 – 6000 Gb
Paired end reads per run 1.6B – 40B
Max read length 2 × 250 bp
Run time 13 – 44 hours
2 lanes, up to 4 channels per lane
Up to 384 samples per channel
Softwares
NovaSeq Control Software v1.7.5
Sequencing Analysis Viewer v2.4.7
bcl2fastq v2.20.0.422

4200 TapeStation system
The Agilent 4200 TapeStation system is an established automated electrophoresis tool for DNA and RNA sample quality control. Fully automated sample processing enables the unattended analysis of size, concentration and integrity. The TapeStation system provides a complete solution for true end-to-end sample quality control within any next-generation sequencing (NGS) or Biobank workflow. This covers the full DNA and RNA range and offers high levels of flexibility in a simplified workflow with ready-to-use ScreenTapes.
Any sample number between 1 and 96 can be analyzed, with constant costs per sample providing scalable throughput.
Results are obtained quickly in as little as 1 to 2 minutes per sample, or less than 90 minutes for 96 samples.
Achieve user-independent results with minimal manual intervention and excellent reproducibility for sizing, concentration and integrity assessment.
Requires as little as 1 to 2 µL of DNA or RNA samples, even for high sensitivity analysis.
Softwares
Agilent TapeStation Software v3.2

Biomek i7 Hybrid liquid handling automation systems
From Beckman Coulter, two systems are present: A – NGS library preparation instrument and B – Nucleic acid extraction instrument.
The systems allow the extraction of DNA and RNA from biological samples and the processing of nucleic acid samples for NGS library preparation, quantification, normalization and pooling for sequencing. The system can transfer liquids on 96well or 384well plates. It allows the quantitation and evaluation of DNA/RNA samples using measurements in absorbance and fluorescence. A FlyBy barcode reader is present for samples and reagents traceability. Control and security systems include: two cams for remote control of the deck, a light curtain for system freezing in case of external interferences, and a cabinet enclosure for sample protection.
Different third part protocols can be implemented on the instruments, as well as new protocols for customized applications. The system can transfer liquids on 96well or 384well plates using a 96 positions multichannel pipetting head (1-300 µl in A, 5-1200 µl in B) or a span-8 system (1-1000µl). The working area is composed of 26 positions for consumables, 8 positions for multichannel tips loading and the integrated devices. Consumables can be moved on the deck using two independent grippers. Samples and reagents can be shaked or incubated at fixed temperature using the following integrated devices: orbital shaker for different kind of labware, static peltier (one in B and two in A) and shacking peltier for 96 well plates and thermoblock (usually used for 1.5 ml vials). Two wash stations are present for tips washing and recycling from 96 multichannel head and span-8 system. An integrated thermocycler (ATC) is present for sample incubation and on-deck PCR (only in A). In the left side module a BMG FLUOstar Omega spectrofluorometer has been installed for integrated quality control steps.
Softwares
Biomek Software v5.1.10
BMG Omega Package v5.70 Edition 4
ATC v1.3.

Contact:
Dr. Danilo Licastro  (tel:)