MASSIVE PARALLEL SEQUENCING
ABOUT
Massive Parallel Sequencing (MPS), also known as Next Generation Sequencing (NGS) is the collection of technologies that are able to genotype (determine the sequence) of DNA or RNA fragments in a massive and parallel fashion.
The second generation MPS technologies rely on clonal amplification of the DNA/RNA fragments to be genotyped, whilst third generation MPS technologies can directly sequence single DNA/RNA fragments without prior clonal amplification.
The second generation technologies generate short sequences of the clonally amplified fragments and are referred to as short-read sequencers. They rely on sequencing-by-synthesis (SBS), where the stepwise incorporation of nucleotides - starting from a sequencing primer - is detected through changes in either fluorescence, pH or impedance. These technologies generally have a low error rate.
The third generation technologies generate long sequences and are referred to as long-read sequencers. The sequence of the DNA (or RNA) fragment to be inferred, is established by measuring either (1) the potential difference over a membrane when this fragment migrates through an nanopore embedded in this membrane or (2) the real-time incorporation of fluorescently labelled nucleic acids by a polymerase. Generally, these technologies have a higher error rate than the 2nd generation sequencing technologies.
METHODOLOGY
There are currently 2 mainstream methods offered by BRIGHTcore : (1) a short-read sequencing technology using the Illumina Sequencing By Synthesis (SBS) method and (2) a long-read sequencing technology using the Oxford Nanopore Technologies (ONT) method.
The library preparation for both technologies rely on similar principles : (1) get your DNA (or RNA/cDNA) to a specific fragment size and (2) modify this DNA so it is flanked by an adapter sequence for the sequencer to 'recognize'. More information on these basic steps can be found here :
The sequencing principle of either the Illumina as Oxford Nanopore technologies is visualized in the movies below. The specifications and applications are listed in the chapters below.
INSTRUMENTS
The following devices are available within our portfolio for Massive Parallel Sequencing.
Illumina MiSeq
Low capacity short read sequencer. Genotypes clonally amplified library fragments through sequencing-by-synthesis (SBS).
Illumina NovaSeq 6000
High capacity short read sequencer. Genotypes clonally amplified library fragments through sequencing-by-synthesis (SBS).
NanoPore GridION x5
Long read sequencer. Types single molecules (3rd generation) via steric disturbance of ionic flow through nanopore embedded in membrane.
SPECIFICATIONS
The specifications are subdivided in 2 sections : (1) the data quantity and (2) the quality and applications.
More information can be found on the supplier page through the corresponding buttons in the 'Data quantity' section.
Data quantity
Instrument
Flow cell types
Max. read length
Max. capacity (bases)
Max. capacity (reads)*
Max. run time
* Abbreviations used : SR = Single Read ; PE = Paired End ; M = million ; B = Billion ; Gb = Gigabases
Quality and applications
Instrument
Applications
Quality**
Remarks
Illumina MiSeq
Small genomes, Amplicon resequencing, Mitochondrial resequencing
> 80% bases higher than Q30 at 2 × 150 bp
> 75% bases higher than Q30 at 2 × 250 bp
> 70% bases higher than Q30 at 2 × 300 bp
As the lowest capacity short read instrument, it unfortunately also comes at the highest cost/base. The possibility to generate high quality 'long' reads (up to 300 bp) is the main advantage.
The MiSeq can only sequence 1 flow cell per run.
Illumina NovaSeq 6000
Whole Genome Sequencing, Whole Exome Sequencing, Large Gene Panels, RNAseq, Methyl-seq, ChIPseq, Small genomes, Mitochondrial resequencing
≥ 90% of bases higher than Q30 at 2 × 50 bp
≥ 85% of bases higher than Q30 at 2 × 100 bp
≥ 85% of bases higher than Q30 at 2 × 150 bp
≥ 75% of bases higher than Q30 at 2 × 250 bp
As the highest capacity short read instrument, it is the cheapest/base. Whole Human Genomes at 30x coverage are possible at prices around 1000€ (S4 flow cell). It's ability to sequence 250bp reads (SP flow cell) is also a big plus.
The NovaSeq can sequence 2 flow cells simultaneously.
NanoPore GridION x5
Small genomes, Long Range Amplicon sequencing, RNAseq, Methyl-seq, Whole Genome Sequencing
The quality is highly dependent on (1) flow cell type (2) sequencing kit and (3) basecaller used.
- FC R9.4.1 and kit 10 : avg. Q-score = ca. 12 (Q12)
- FC R10.4 and kit 14 : avg. Q-score = ca. 20 (Q20)
The ability to generate long reads and the flexibility of the run times are highly useful in (small) genome sequencing and metagenomics. This, despite its higher error rate. Single contig small genomes are possible. Also, due to the short run times, the Nanopore technology proved to be essential in Sars-Cov2 typing.
The GridION can sequence 5 flow cells simultaneously.
** To know more about the Q score metric, please CLICK HERE
CERTIFICATES
Part of accredited process
Sciensano recognized
245-MED (Hopital Erasme)
141-MED (UZ Brussel)
EFI
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CONTACTS
BRIGHTcore general inquires
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UZ Brussel
BRIGHTcore
Laarbeeklaan 101
1090 Brussels
SAMPLE TYPES
We aim to keep the list with sample types updated. However, if you believe we offer this test on other sample types, or if you have a very specific sample type you'd like to evaluate : please contact us.
To have a more detailed overview, please consult the Application of interest.
Tissue | Recipient | Quantity | Transport | Purpose / Extract |
|---|---|---|---|---|
Sars-Cov2 on UTM (naso/oropharyngeal) | UTM (Universal Transport Medium) or PBS tube w/o inactivation buffer | 500 µl (Ct<=25) | Room temperature | RNA |
RNA from various tissues | Microtube* or 96 well plate | See test details/specifications | Ice (max. 20 min) or dry ice | RNA |
DNA from various tissues | Microtube* or 96 well plate | See test details/specifications | Room temperature | DNA |
NGS library (non-PCR free) | Microtube* or 96 well plate | See test details/specifications | Room temperature or ice pack | DNA |
NGS library (PCR free) | Microtube* or 96 well plate | See test details/specifications | Ice (max. 1 day) or dry ice | DNA |
PCR products (amplicons) | Microtube* or 96 well plate | See test details/specifications | Room temperature | DNA |
Sars-Cov2 RNA (naso/oropharyngeal) | Microtube* or 96 wel plate (plate is preferred) | Log(copies/ml) > 3 (min. 10 µl) | Ice (max. 20 min) or dry ice | RNA |
Tissue of various origins | Cryovial containing stabilizer (eg. RNAlater) or tissue homogenized on lysis buffer | Application/tissue specific (contact us) | Dry ice | RNA |
FFPE section of various tissues | Microtube* | Min. 1x 10 µm section (up to 2 mm³ tissue) ; Tumor load preferentially >10% | Room temperature | RNA |
FFPE block of various tissues | Paraffin block (in cassette) | Representative block ; Tumor load preferentially >10% ; To be processed by our pathology department (microtome section needed) | Room temperature | RNA |
Blood | DBS / Guthrie card / Punch in microtube* | Min. 1 punch | Room temperature | DNA |
Blood | Streck RNA Complete BCT | 10 ml | Room temperature | cfRNA/exoRNA |
FFPE section of various tissues | Microtube* | Min. 1x 10 µm section (up to 2 mm³ tissue) ; Tumor load preferentially >10% | Room temperature | DNA |
Cells of various tissue origins | Microtube* with cells on lysis buffer | > 5 million cells | Dry ice | RNA |
Cells of various tissue origins | Microtube* with cell pellet or on lysis buffer | > 5 million cells | Dry ice | DNA |
Cells of various tissue origins | Culture dish/flask (on medium) | Viable cells | Room temperature | Culture (DNA/RNA) |
Tissue of various origins | Cryovial containing stabilizer (eg. AllProtect) or tissue homogenized on lysis buffer | Application/tissue specific (contact us) | Dry ice | DNA |
Blood | PAXgene Blood RNA tube | 10 ml (2,5 ml blood) | Ice pack | RNA |
Plasma | DNA LoBind 5 ml tube | > 2ml | Dry ice | cfRNA/exoRNA |
Plasma | DNA LoBind 5 ml tube | > 2ml | Dry ice | cfDNA/ctDNA |
Blood | Streck Cell-Free DNA BCT | 9 ml | Room temperature | cfDNA/ctDNA |
FFPE block of various tissues | Paraffin block (in cassette) | Representative block ; Tumor load preferentially >10% ; To be processed by our pathology department (microtome section needed) | Room temperature | DNA |
RNA from various tissues | Microtube* | See test details/specifications | Ice (max. 20 min) or dry ice | RNA |
DNA from various tissues | Microtube* | See test details/specifications | Room temperature | DNA |
Blood | EDTA tube | 10 ml (min. 3 ml) | Room temperature | DNA |
* Microtube can be a either a cryovial or eppendorf tube (0,5 ; 1,5 or 2 ml). Please consider the quality of the tube used (nuclease free ; free of RNA/DNA ; sterile ; LoBind) according to application needed.
DELIVERABLES
Which data files can be retrieved from us for run reservations on our sequencers?
More specific deliverables are available dependent on the Application or requested Bioinformatics support.
Deliverable | Description |
|---|---|
Raw data - bam file | Binary data file containing the aligned reads |
Raw data - fast5 file | Raw data file generated by Oxford NanoPore sequencers |
Raw data - fastq file | Data file containing the raw (unaligned) reads |
Run folder | The complete run folder can be transferred if it concerns a run reservation (private run) |
PLAN EXPERIMENT
There are 2 options available. See below for more details.
RUN RESERVATION
Have full control and produce your own libraries. We will sequence them for you.
To start a reservation you need to get in touch with us to (1) clarify your needs (2) get a specific quote and (3) deliver your libraries.
APPLICATION
Choose a specific application from our portfolio and let us process your samples
Check our applications below. You will find more information on the application pages. Get in touch with us if you don't find what you need.
