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Next Generation Sequencing: Application to
Transfusion Medicine and Immunohematology ?
O. Preynat-Seauve
Laboratory of immunohematology
Hematology Unit
Laboratory medicine unit
Geneva University Hospital
[email protected]
DNA and RNA sequencing
“the process of determining the precise order
of nucleotides within a nucleic acid molecule”
DNA
RNA
Plants
Microbes
Human/animal cells and tissues
Vaccines
...
Blood products
...
The history of sequencing
1977: « Maxam Gilbert
Sequencing »
2013: « next generation methods »
or « high throughput sequencing »
>500 000 sequencing
operations can be runned
in parrallel
WHOLE genome,
transcriptome
, miRNome
etc.
Only fragments
Next Generation Sequencing (NGS):
various methods
Method
Single-molecule
real-time
sequencing
(Pacific Bio)
Ion
semiconductor
(Ion Torrent
sequencing)
Pyrosequencing
(454)
Sequencing by
synthesis
(Illumina)
Sequencing by
ligation (SOLiD
sequencing)
Chain
termination
(Sanger
sequencing)
Read length
5,000 bp average;
maximum read
length ~22,000
bases
200 bp
700 bp
50 to 250 bp
50+35 or 50+50
bp
400 to 900 bp
98%
99.9%
98%
99.9%
99.9%
up to 5 million
1 million
up to 3 billion
1.2 to 1.4 billion
N/A
Accuracy
Reads per run
99.999%
consensus
accuracy; 87%
single-read
accuracy
50,000 per SMRT
cell, or ~400
megabases
Time per run
30 minutes to 2
hours
2 hours
24 hours
1 to 10 days,
1 to 2 weeks
20 minutes to 3
hours
Advantages
Longest read
length. Fast.
Detects 4mC,
5mC, 6mA.
Less expensive
equipment. Fast.
Long read size.
Fast.
Potential for high
sequence yield,
and desired
application.
Low cost per
base.
Long individual
reads. Useful for
many
applications.
Homopolymer
errors.
Runs are
expensive.
Homopolymer
errors.
More expensive
and impractical
Equipment can be Slower than other
for larger
very expensive.
methods.
sequencing
projects.
Moderate
throughput.
Disadvantages
Equipment can be
very expensive.
The most widely used system is provided by
the Illumina company
“the simultaneous sequencing of millions of tiny fragments of DNA on
the surface of a glass slide about the size of a large matchbox”
The machine produces millions of short sequences called « READS »
Millions of reads
ATGG...CGCA
TTGA...ATGCG
TATA....CTA
GGC...AATAA
etc.
etc.
Reads (= fragments) are reasembled by
softwares into « CONTIGS »
TTGA...ATGCGGGC...AATAAATGG...CGCA
CONTIGS are identified using databases
(bioinformatics)
each portion of the genome/RNome is represented
multiple times in different fragment frames
(fragmentation is at random)
Genome position
Whole genome/transcriptome
sequencing: interest for
immunohematology and transfusion
medicine ?
Whole sequencing for
immunohematology ?
Single analysis of the entire blood groups genotype
 Determination of a global profile in one step
 Exhaustive identification of blood groups variants, rare
genotypes etc.
Targets ? Blood groups antigens, HLA, minor antigens
* Too heavy /expensive/slow as compared to existing
methods?
* Less quantitative than PCR ?
* Sensitivity ?
* False positive/false negative rates? (and controls for each
gene!)
* Can we easily deduce the phenotype from the genotype ?
To technically sequence a whole genome is currently « easy »
and not to much expensive
… and finally you obtain a CD with millions and millions of data
Remark: do not start if you do not have in your team a
bioinformatician!
sequencing (2 weeks)
analysis (months, years!)
Interest of sequencing for
transfusion medicine?
Landscape of nucleic acids present in blood products ?
 The complete nucleic acid content in blood products is not known
Blood product
Nucleic acids associated
with residual leukocytes
Cell-free nucleic acids
Nucleic acid associated
with cells (red blood cells
or platelets)
Landscape of non-human nucleic acids in blood products ?
All the viruses that « escape » to blood products qualification:
• Emergent viruses ?
• Inocuous viruses (that could have impact on immunocompromised patients)
• Other infectious agents signatures ?
Fresh frozen plasma
Red blood cells concentrate
Platelets concentrate
Reinforcment (or not) of pathogens inactivation ?
Additonal virus testing for immunocompromised patients ?
Development of a bioinformatic software for virus
screen in a whole RNA sequence (Illumina)
Specificity
Pos. controls
Assemblies
(CONTIGS)
Dr Thomas Petty, postdoc
Pipeline validation using CMV/Sendaï
virus-infected cells
Dr Erika Cosset, postdoc
Dr Thomas Petty, postdoc
neg. control
Virus-free
samples
(glioblastoma)
neuroepithelial cells
neuroepithelial cells+CMV
neuroepithelial cells+Sendaï
virus
Percent of the virus genome
that is covered by reads =
GENOME COVERAGE
Number of
matching
reads
This binary computational analysis mixing genome coverage and number of reads
provide useful informations in this context of virus discovery
Low virus replication
High virus replication
Latent
No virions/viral gene
reactivation
Latent viruses reactivating some
genes without virions replication
(CMV)
Ongoing project: virus screen in blood
products
10 pools of 10 plasma unit samples ( 100 donors)
10 pools of 10 red blood cells unit samples ( 100 donors)
Negative controls (buffer alone)
Positive controls: blood products samples infected by CMV/Sendaï virus
DNA seq
RNA seq
Bioinformatic pipeline
Exhaustive « picture » of the virological status of blood products
Landscape of human nucleic
acids in blood products ?
CELLS
mRNA (haemoglobin !)
rRNA
tRNA
miRNA
residual plasma
mitDNA
Cell-free nucleic acids
plasma
Cell-free nucleic acids
Residual leukocytes
Genomic/mitochondrial DNA
all RNAs
Microparticles
miRNA
Red blood cells
plasma
platelets
Cell-free nucleic acids (plasma)
ds short DNA (70-200 base pair)
ds long DNA (< 21 kb)
mRNA
miRNA (very active !)
Neutrophil Extracellular Traps (NETs)
Sources: cell necrosis, apoptosis, active secretion (lymphocytes,
neutrophils)
Nucleic acids present in microparticles
BIOLOGICAL ACTIVITY IN RECIPIENT ?
NGS and transfusion: concluding remarks
 research: provide a new tool to improve the knowledge of
transfusion and immmunohematology
 routine: Potential interest in the future ??
Laboratory of immunohematology
Geneva University Hospital
Erika Cosset
Thomas Petty
Olivier Preynat-Seauve
Blood Transfusion Center
Geneva University Hospital
Emanuel Rigal
Soraya El-Dusouqui
Hematology Unit
Geneva University Hospital
Thomas-Pierre Lecompte
ARTERES Foundation, Geneva
ISREC Foundation, Lausanne
Egon Naef Foundation, Geneva
Department of Genetic and Laboratory Medicine
Laboratory of Virology
Geneva University Hospital
Laurent Kaiser
Samuel Cordey
Oncology Unit
Geneva University Hospital
Pierre-Yves Dietrich
Valérie Dutoit
Swiss Institute of Bioinformatic
Evgeny Zbodnov
Ismel Palladieau
Genomic Core Facility
Faculty of medicine
Geneva
FASTERIS SA, Plan-Les-Ouates