next generation sequencing: application to transfusion medicine and immunohematology ? o....
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Next Generation Sequencing: Application to Transfusion Medicine and Immunohematology ?
O. Preynat-SeauveLaboratory of immunohematologyHematology UnitLaboratory medicine unitGeneva University [email protected]
DNA and RNA sequencing
“the process of determining the precise order of nucleotides within a nucleic acid molecule”
DNARNA
PlantsMicrobesHuman/animal cells and tissuesVaccines...Blood products...
The history of sequencing1977: « Maxam Gilbert Sequencing »
2013: « next generation methods » or « high throughput sequencing »
Only fragments
WHOLE genome, transcriptome, miRNome etc.
>500 000 sequencing operations can be runned in parrallel
Next Generation Sequencing (NGS): various methods
MethodSingle-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 length5,000 bp average;
maximum read length ~22,000
bases200 bp 700 bp 50 to 250 bp 50+35 or 50+50
bp 400 to 900 bp
Accuracy
99.999% consensus
accuracy; 87% single-read
accuracy
98% 99.9% 98% 99.9% 99.9%
Reads per run50,000 per SMRT
cell, or ~400 megabases
up to 5 million 1 million up to 3 billion 1.2 to 1.4 billion N/A
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
AdvantagesLongest 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.
DisadvantagesModerate
throughput. Equipment can be
very expensive.
Homopolymer errors.
Runs are expensive.
Homopolymer errors.
Equipment can be very expensive.
Slower than other methods.
More expensive and impractical
for larger sequencing
projects.
“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 most widely used system is provided by the Illumina company
The machine produces millions of short sequences called « READS »
Millions of readsATGG...CGCATTGA...ATGCGTATA....CTAGGC...AATAAetc.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 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
Nucleic acid associated with cells (red blood cells or platelets)
Cell-free nucleic acids
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)
Dr Thomas Petty, postdoc
Assemblies(CONTIGS)
Specificity
Pos. controls
Pipeline validation using CMV/Sendaï virus-infected cells
Dr Thomas Petty, postdocDr Erika Cosset, postdoc
neg. control
Virus-free sam
ples (glioblastom
a)
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
Latent
Latent viruses reactivating some genes without virions replication (CMV)
High virus replication
No virions/viral gene reactivation
Low virus replication
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
CELLSmRNA (haemoglobin !)rRNAtRNAmiRNAmitDNA
residual plasmaCell-free nucleic acids
Residual leukocytesGenomic/mitochondrial DNAall RNAs
Red blood cells plasma platelets
MicroparticlesmiRNA
plasmaCell-free nucleic acids
Cell-free nucleic acids (plasma)
ds short DNA (70-200 base pair)ds long DNA (< 21 kb)mRNAmiRNA (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 immunohematologyGeneva University Hospital
Erika CossetThomas PettyOlivier Preynat-Seauve
Blood Transfusion CenterGeneva University Hospital
Emanuel RigalSoraya El-Dusouqui
Laboratory of VirologyGeneva University HospitalLaurent KaiserSamuel Cordey
Oncology UnitGeneva University HospitalPierre-Yves DietrichValérie Dutoit
Swiss Institute of BioinformaticEvgeny ZbodnovIsmel Palladieau
Genomic Core FacilityFaculty of medicineGeneva
FASTERIS SA, Plan-Les-Ouates
ARTERES Foundation, GenevaISREC Foundation, LausanneEgon Naef Foundation, GenevaDepartment of Genetic and Laboratory Medicine
Hematology UnitGeneva University Hospital
Thomas-Pierre Lecompte