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 mainstraim 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

Illumina MiSeq

Standard v2
Standard v3
Nano v2
Micro v2

250 bp
300 bp
250 bp
150 bp

39h
56h
28h
19h

8,5 Gb
15 Gb
0,5 Gb
1,2 Gb

15M SE - 30M PE
25M SE - 50M PE
1M SE - 2M PE
4M SE - 8M PE

Illumina NovaSeq 6000

SP
S1
S2
S4

250 bp
150 bp
150 bp
150 bp

38h
25h
36h
44h

400 Gb
500 Gb
1250 Gb
3000 Gb

0,8B SE - 1,6B PE
1,6B SE - 3,2B PE
4,1B SE - 8,2B PE
10B SE - 20B PE

NanoPore GridION x5

R9.4.1
R10.4

> 4 Mb

72h (interruptible at all time)

< 50 Gb

< 5M (for avg. 10K bp reads)

* 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 usefull 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

 
BELAC
Part of accredited process
Sciensano
Sciensano recognized
BELAC
245-MED (Hopital Erasme)
BELAC
141-MED (UZ Brussel)
EFI
EFI

DOWNLOADS

 
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Analyses Génomiques Experts FR v2

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Genoom Testing Niet-expert NL v2

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Analyses Génomiques Non-experts FR v2

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Genoomonderzoek in de genetica Expert NL v2

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Genome-wide genetic testing Expert EN v2

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Genome-wide genetic testing Non-expert EN v2

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CONTACTS

 
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BRIGHTcore general inquires

+32 (0)2 477 64 79 (sec)

UZ Brussel

BRIGHTcore

Laarbeeklaan 101

1090 Brussels

SAMPLE TYPES

 

We aim to keep te list with sample types updated. However, if you believe we offer this test on other sample types, of 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.

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Tissue
Recipient
Quantity
Transport
Purpose / Extract
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. 5 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.

Image by Alfred Quartey
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.

Image by David Švihovec

APPLICATIONS

 

The following applications are readily available for you to make use of. Please be aware that some of those applications are multi-purpose and more can be found on the corresponding application page.  If you don't find what you need, please contact us!