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The NGS Core Facility is running two different NGS technologies, each of which have advantges/disadvantages depending on the application. Specifically, three different NGS platforms including the Illumina NextSeq 550, an Illumina MiSeq as well as an IonTorrent PGM System are being used. In order to allow single cell sequencing, the lab runs a 10xGenomics platform that uses droplet technology to generate single cell suspensions. All platforms are run as operator-based systems.
Illumina NextSeq 550
The Illumina NextSeq® 550 is the largest desktop next-generation sequencing (NGS) system from Illumina capable of sequencing a 30x human genome in a single run. Two different flow cell options and multiple reagent configurations are providing a maximum application flexibility (130 to 400M clusters; 40 to 120 Gb).
Accordingly, the system is suitable for a high number of different applications, e.g. whole exome- or genome sequencing, RNAseq, targeted resequencing or gene expression profiling and single-cell sequencing. The sequencing technology is based surface-based bridge amplification for clonal amplification and sequencing of each generated cluster using the Illumina "sequencing by synthesis" (SBS) method with reversible terminator chemistry.
Features
- Scalability (20–120 Gb) in a single run supporting a broad range of applications and study sizes
- Sequencing runs, including on-board cluster generation, complete in 12–30 hours
- Proven SBS chemistry with single-base extension enables accurate sequencing of homopolymers (sometimes a problem on the IonTorrent system)
- Fully automated paired-end sequencing
- Automated BeadChip array scanning and image file generation
- Approximately 7000 peer-reviewed publications have been published using Illumina SBS sequence data, and more than 24,000 peer-reviewed publications have been published using Illumina array technology
- Number of reads per run
- preadjustment of library diversity is required for optimum sequence quality
Data Generation
Throughput per run
- Up to 400 million reads per run (eg, clusters passing filter) using High Output flow cell
- Up to 130 million reads per run (eg, clusters passing filter) using Mid Output flow cell
Sequencing flexibility
- 100–120 Gb data per 2 × 150 bp run using High Output flow cell and reagents
- 33–40 Gb data per 2 × 150 bp run using Mid Output flow cell and reagents
- Set-up options include single-read or paired-end runs
Array Scanning
- Flow cell options (eg, Mid or High Output flow cells) can be used to select data output levels
- Read length is fully adjustable up to 300 base pairs
- Single array imaged per scanning session
- Up to 12 samples imaged per run with current array support
Instrumentation
Illumination
- 12 light-emitting diodes at 520 nm, 650 nm
Instrument Control Computer
- Dual Intel Xeon E5-2448L 1.8 GHz CPU with 96 GB of RAM included for instrument control, processing images, and base calling
- Conducts real-time analysis processing that automatically produces image intensities and quality-scored base calls directly on the instrument computer
Sequencing run time
- ~ 12 hours for a 1 × 75 bp single-read sequencing run
- ~ 18 hours for a 2 × 75 bp paired-read sequencing run
- ~ 30 hours for a 2 × 150 bp paired-read sequencing run
Illumina MiSeq System
The Illumina MiSeq™ System is a desktop next-generation sequencing (NGS) system from Illumina allowing fast and simple targeted or small genome sequencing. The main applications include targeted resequencing, metagenomic analysis and small whole-genome sequencing.
MiSeq reagents enable up to 15 Gb of output with 25 million sequencing reads and 2 x 300 bp read length. The sequencing technology is based surface-based bridge amplification for clonal amplification and sequencing of each generated cluster using the Illumina "sequencing by synthesis" (SBS) method with reversible.
Data Generation
Number of reads per run
- Up to 25 million reads per run (eg, clusters passing filter)
Throughput per run
- 25 GB data per 2 × 300 bp run (depending on the kit)
Instrumentation
Illumination
- 12 light-emitting diodes at 520 nm, 650 nm
Instrument Control Computer
- Base Unit: Intel Core i7-2710QE 2.10 GHz, 16 GB RAM Memory, 750 GB Hard Drive
Sequencing run time
- ~ 5.5 hours for a 2 × 25 bp paired-read sequencing run using MiSeq Reagent Kit v2
- ~ 24 hours for a 2 × 150 bp paired-read sequencing run using MiSeq Reagent Kit v2
- ~ 39 hours for a 2 × 250 bp paired-read sequencing run using MiSeq Reagent Kit v2
- ~ 21 hours for a 2 × 75 bp paired-read sequencing run using MiSeq Reagent Kit v3
- ~ 56 hours for a 2 × 300 bp paired-read sequencing run using MiSeq Reagent Kit v3
- ~ 19 hours for a 2 × 150 bp paired-read sequencing run using MiSeq Reagent Kit v2 Micro
- ~ 17 hours for a 2 × 150 bp paired-read sequencing run using MiSeq Reagent Kit v2 Nano
- ~ 28 hours for a 2 × 250 bp paired-read sequencing run using MiSeq Reagent Kit v2 Nano
Ion Torrent PGM
The DNA-Sequencing on the desktop NGS system PGM from Ion Torrent Life Technologies is based on the principle of Semiconductor Sequencing developed in 2007. This technology allows a real-time measurement of hydrogen ions produced during DNA replication and thereby directly translates genetic information (DNA) to digital information (DNA sequence).
This is enabled by an ion semiconductor chip on which the sequencing reaction takes place. The different available chips allow a flexible output for ultimate project flexibility. Additonally, the sequencing times are short, e.g. 30 min for a 1 x 35 bp on a 314 chip or 8 hours for a 1 x 400 bp run on a 318 chip.
Data Generation
Number of reads per run
- Up to 0.5 million reads per Ion 314 chip (eg, reads passing filter)
- Up to 3 million reads per Ion 316 chip (eg, reads passing filter)
- Up to 5.5 million reads per Ion 318 chip (eg, reads passing filter)
Throughput per run
- 30-50 Mb per 1 x 200 bp reads and 60-100 Mb per 1 x 400bp read on a Ion 314 Chip
- 300-600 Mb per 1 x 200 bp reads and 600 Mb - 1 Gb per 1 x 400bp read on a Ion 316 Chip
- 600 Mb - 1 Gb per 1 x 200 bp reads and 1.2 - 2 Gb per 1 x 400bp read on a Ion 318 Chip
Instrumentation
Configuration
A single free-standing tower computer appliance, included with the purchase of the Ion PGM™ System. Includes Torrent Suite™ Software with all necessary software components to deliver signal processing, base calling, read alignment, and variant calling.
Instrument Control Computer
- Dual 6-core 2.66 GHz CPUs, 48 GB RAM, 16 TB Storage
Sequencing run time
- ~ 2 hours per 1 x 200 bp reads and ~ 4 hours per 1 x 400bp read on a Ion 314 Chip
- ~ 3 hours per 1 x 200 bp reads and ~ 5 hours per 1 x 400bp read on a Ion 316 Chip
- ~ 4.5 hours per 1 x 200 bp reads and ~ 7 hours per 1 x 400bp read on a Ion 318 Chip
10xGenomics Chromium Controller
The Chromium Controller allows for high-throughput single cell transcriptome, immunome, or copy number analysis in a variety of cell types as well as single-cell nuclei. The flexible workflow encapsulates single cells, nuclei or DNA together with gel beads into nanodroplets. The underlying GemCodeTechnology enables the execution of thousands of micro-reactions in parallel. A GEM is a “Gel bead in EMulsion” droplet that encapsulates each micro-reaction within the Chromium System. 10x genomics offers a multitude of kit solutions for single cell 3’ and 5’ transcriptome, single cell copy number variation, single cell immune receptor profiling and single cell epigenetic analyses. Moreover, the Chromium Controller can be utilized for sequencing-ready library generation of whole genome or exome analyses.
Features
- Small benchtop instrument (20x20x30cm)
- Touch screen interface and retractable drawer
- Fast workflow from cell suspension to sequencing-ready library
- Captures 100-10.000+ cells per sample and up to 80.000 cells per run in < 7 minutes
- Recovers up to ~65 % of cells (typically 5 0%)
- Low doublet rate (~0.9 % per 1,000 cells)
- Compatible with Illumina HiSeq4000, NextSeq and MiSeq sequencers
- The cell size limit is comparatively high (up to 50 µm).