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Genética: las técnicas del
laboratorio en secuenciación
tumoral: ¿en qué consiste el
análisis y qué información debe
constar en el informe de
resultados?
Ana Vivancos Vall d’Hebron Institute of Oncology
Clinical
approach
Pathology
approach
Molecular
approach
NextSeq (Illumina) Up to 400 millon sequences per day, 120 Gb data MiSeq (Illumina) Up to 25 millon sequences per day, 15 Gb data
2000
ABIPrism (Applied Biosystems)
Up to 2304 per day (96 sequences per hour)
1 haploid human genome 3 Gb
2017
NGS workflow
Library
preparation Sequencing Data analysis
Whole
genome seq
Capture-seq
(Exome-seq)
Amplicon-
seq
mRNA-seq
Material DNA DNA DNA RNA
Chemistry Fragment &
sequence
Fragment, enrich specific
regions & sequence
PCR amplify
regions os
interest &
sequence
Fragment, RT &
sequence
Point mutations & indels X X X X
CNA (a 2n control is required) X X
Rearrangements / Gene Fusions X * (only if they take place
in enriched regions)
X
How to? Mutation detection: high local coverage
EGFR L858R (8%) 6
mutant reads/ 68 total
reads
Alteration Whole
genome
Capture-seq
(Exome-seq)
Amplicon
Point mutations & indels X X X
How to? Copy Number Alterations: wide and homogeneous coverage
Shallow WGS: whole genome, very low coverage 5X
Whole
genome seq
Capture-seq
(Exome-seq)
Amplicon-
seq
mRNA-seq
CNA (a 2n control is required) X X
How to? Gene Fusions detection, much simpler at the RNA level
Whole
genome seq
Capture-seq
(Exome-seq)
Amplicon-
seq
mRNA-seq
Rearrangements / Gene Fusions X * (only if they take place
in enriched regions)
X
T
N
Gene-panel capture approaches (up to 24000 genes)
Exome-seq (24000 genes)
Whole genome sequencing
Amplicon-seq (up to 200 genes)
The larger the panel, the higher the need of including a normal sample
T
N
NGS IMPORTANT TIPS
• CAN’T DO EVERYTHING WITH A SINGLE APPLICATION
• DEPENDING ON WHAT WE WANT TO STUDY, A CAREFUL PLANNING IS
REQUIRED, TAKING INTO ACCOUNT: SPECIMEN, SAMPLE PREPARATION,
COVERAGE REQUIREMENTS
• BE AWARE OF LIMITATIONS AND BIASES OF OUR EXPERIMENT
Reporting…
MDs training on how to
interpret variants
Reporting…
MDs should be trained on
understanding the test and
its limitations
Reporting…
There is no reference
classification system for
variants, so might differ from
lab to lab
Reporting…
Regions sequenced with the
panel, it’s not the whole
genome!
Reporting…
Regions that, although in the
panel, were NOT sequenced
successfully in the particular
sample (mainly because of
FFPE quality issues)
A mutation in those regions
WILL NOT be detected
N (2n): stroma, lymphcytes,
normal surrounding tissue
Reporting MAFs?
TUMOR CELLS
Mutations may be present in all tumor cells:
CLONAL
OR
May be present in a subset of cells:
SUBCLONAL. In some instances, resistance
mechanisms… ex. EGFR T790M
Mutated allele
Some tumor types are
stroma enriched, ex.
pancreas
BRCA1:NM_007294:exon2:
c.60_69del:p.K20fs
+ LOH…
MAFs depend on:
Tumor purity/ Stroma content
Clonality of variant
Copy number of the locus
LOH status of the locus
VUS… Germline variants…
Actionability or prognosis impact
Ddbb…
Other..
Thanks!!!!