Next Generation Sequencing: Application to Transfusion Medicine and Immunohematology ?

O. Preynat-SeauveLaboratory of immunohematologyHematology UnitLaboratory medicine unitGeneva University Hospitalolivier.preynat-seauve@hcuge.ch

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 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

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

Landscape of human nucleic acids in 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