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!!!!