Bioinformatica e genomica comparata: nuove strategie sperimentali e computazionali per la produzione e analisi di dati NGS finalizzati a sviluppare processi e prodotti innovativi per la salute dell’uomo, l’ambiente e l’agroalimentare.

1. Bioinformatica e genomica comparata: nuove strategie
sperimentali e computazionali per la produzione e
analisi di dati NGS finalizzati a sviluppare processi e
prodotti innovativi per la salute dell’uomo, l’ambiente e
l’agroalimentare.
Ernesto Picardi
University of Bari
IBBE-CNR
http://www.pesolelab.it/
ernesto.picardi@uniba.it
BiP-­‐Day
5/12/2013

2. Next-­‐Genera*on
Sequencing
Several NGS platforms are now available on the market, which are progressively
improved in term of throughput and cost- and time-efficiency.
Ion Proton
Roche / 454 Genome
Sequencer FLX titanium
(800 bp, 800 Mb / run)
PacBio
Illumina / Solexa
Genetic Analyzer HiSeq 2000
(150x2 bp, 600 Gb / run)
Applied Biosystems
SOLiD 4 SystemTM
(100x2 bp, 400 Gb / run)

3. Worldwide
distribu*on
of
NGS
pla;orms
>2558
total
machines
(>1000
US,
>300
China,
..,
>50
Italy)
Source:
omicsmap.com

4. Our
first
experience
RNA-­‐Seq
reads:
-­‐ Roche
454
(GS-­‐20):
>120000
conBgs
(assembled
reads
from
4
Bssues)
-­‐ Illumina/Solexa:
>
200
M
reads
(from
2
Bssues)
-­‐ SOLiD
:
>
320
M
reads
(from
4
Bssues)
Jaillon
et
al.
2007
Nature

5. Available
compu*ng
facili*es
at
UNIBA/IBBE-­‐CNR
HP
Proliant
Server
-­‐ 256
GB
RAM
-­‐ 40
cores
-­‐ 2
RAIDs
(24
+
36
TB)
HP
Proliant
Server
-­‐ 2
TB
RAM
-­‐ 80
cores
-­‐ 2
RAIDs
(36
+
48
TB)
-­‐ 4
GPUs
Tape
library
3
Xserve
Apple
-­‐ 16x3
GB
RAM
-­‐ RAID
3
TB
External
Partners

6. Available
NGS
pla;orms
at
UNIBA/IBBE
Illumina
MiSeq
-­‐ >
50
M
reads/run
-­‐ Paired
2x300
• DNA sequencing (DNA-Seq)
-­‐
genome
resequencing
(SNPs,
CNV,
GWAS)
-­‐
de
novo
sequencing
-­‐
idenBficaBon
of
genome
structural
variants
(cancer
genome)
-­‐
Epigenomics
(chromaBn
state
and
genome
methylaBon)
-­‐
Metagenomics
(Microbiota
analysis
of
clinical
/environmental
samples)
• RNA sequencing (RNA-Seq)
-­‐
QualitaBve
and
quanBtaBve
analysis
of
the
Transcriptome
-­‐
IdenBficaBon
and
characterizaBon
of
miRNAs
and
other
ncRNAs
-­‐
RNA
ediBng
-­‐
Metatrancriptomics
(funcBonal
analysis
of
environmental
samples)
Illumina
HiSeq1500
-­‐ >
300
GB/run
-­‐ Paired
2x100

7. DNA
sequencing:
variant
analysis
ProducBon
of
DNA-­‐Seq
reads
for
the
idenBficaBon
of
SNPs
or
Indels
in
normal
and/or
pathological
condiBons,
and
development
of
dedicated
socware.
QC
(quality
control)
Align
to
reference
genome
Variant
(SNP
&
INDEL)
calling
Epstein-­‐Barr
genotyping
In
mulBple
sclerosis
(30
samples
–
0.2
M
reads/sample
)
A
“family
trio”
for
variants
linked
to
the
Majewski
syndrome
–
like
disease
(>260
M
reads
Illumina)
Variant
annotation
Functional
validation
Somatic
mutations
in
human
tissues
(3
donors/
6
tissues)
(18
WXSs
>
1080
M
reads
Illumina)

8. DNA
sequencing:
de
novo
sequencing
Using
the
Illumina
MiSeq
plaform
we
have
sequenced
>20
full
prokaryoBc
genomes
generaBng
on
average
2
M
reads
per
sample.
Illumina
MiSeq
Reads
In
collaboraBon
with:
-­‐ Doh.
Parisi
(IsBtuto
ZooprofillaBco)
-­‐ Prof.
Palmieri
(UNIBA-­‐IBBE)
-­‐ Doh.
Ceci
(IBBE)
-­‐ Prof.
Golyshin
(Bangor
Univ.
UK)
Read
Assembling
ConBgs

9. DNA
sequencing:
sequencing
of
mtDNA
Assembly
of
full
mtDNAs
in
human
by
WXS
and
WGS
experiments.
In
collaboraBon
with:
Prof.
Akmonelli
Picardi
and
Pesole
2012
Nature
Methods

10. DNA
sequencing:
metagenomics
Deep
sequencing
of
environmental
samples
to
funcBonally
characterize
the
biodiversity
(no
need
for
laboratory
culBvaBon
and/or
isolaBon
of
individual
specimens).
BioMaS
(BioinformaBc
analysis
of
Metagenomic
ampliconS)
hhp://webgateway.ba.infn.it:9999/
Raw
Data
Merged
and
Denoised
Sequences
Dereplicated
Sequences
DB
matching
TANGO
Specimen
assessment
In
collaboraBon
with:
Prof.
Valiente
(Catalogna
Univ.)
CorrelaBon
between
Invasive
Species
and
the
microbial
composiBon
(>
15
M
reads)
Microbiota
variaBon
in
colorectal
cancer
(>
125
M
reads)
Development
of
BioinformaBc
tools
MICROMAP

11. RNA
sequencing:
RNA-­‐Seq
analysis
Development
of
dedicated
computaBonal
tools
to
automate
the
analysis
of
RNA-­‐Seq
samples.
Transcriptome
variaBons
in
6
ALS
samples
(>120
M
reads
Illumina/sample)
Transcriptome
variaBons
in
6
human
Bssues
(>150
M
reads
Illumina/sample)
Transcriptome
variaBons
in
Alzheimer
disease
(15
samples)
(>150
M
reads
Illumina/sample)

12. RNA
sequencing:
miRNA-­‐Seq
analysis
ProducBon
of
miRNA-­‐Seq
data
and
development
of
ad
hoc
tools
to
analyse
reads
in
mulBple
experimental
condiBons.
miRNA
expression
in
6
ALS
samples
(>5
M
reads
Illumina/sample)
miRNA
expression
in
6
human
Bssues
(>5
M
reads
Illumina/sample)
miRNA
expression
in
aging
and
Alzheimer
disease
(15
samples)
(>3
M
reads
sample)
Dr
David
Horner

13. RNA
sequencing:
RNA
edi*ng
Development
of
the
first
toolkit
to
detect
RNA
ediBng
by
massive
sequencing
data
(RNA-­‐Seq,
DNA-­‐Seq
and
miRNA-­‐Seq).
Picardi and Pesole 2013 Bioinformatics
r1
r2
r3
r4
r5
r1
r2
r3
r4
r5
GGGTGCCTTTATGCAGCAAGGATGCGATATT!
GGGTGTCTTTATGCAGCAAGGATGCGATACTTCGC!
GGGTGCCTTTATGCAGCAAGGATGCGATATTTCG!
GGGTGCCTTTATGCAGCAAGGATGCGATATTTCG!
GGGTGCCTTTATGCAGCAAGGATGCGATATTTCG!
..............A.....................!
TGGGTGCCTTTATGCAGCAAGGATGCGATATTTCGCC gDNA!
..............G.....................!
GGGTGCCTTTATGCGGCAAGGATGCGATATT!
GGGTGTCTTTATGCAGCAAGGATGCGATACTTCGC!
GGGTGCCTTTATGCGGCAAGGATGCGATATTTCG!
GGGTGCCTTTATGCGGCAAGGATGCGATATTTCG!
GGGTGCCTTTATGCGGCAAGGATGCGATATTTCG!
RNA
ediBng
in
ALS
(RNA-­‐Seq:
>120
M
reads/sample
WXS:
>
30
M
reads/sample)
RNA
ediBng
in
human
Bssues
(RNA-­‐Seq:
>120
M
reads/sample
WGS:
>
300
M
reads/sample)
RNA
ediBng
in
Alzheimer
disease
Fluidigm
tech.
and
Illumina
Seq.
(>2000x
per
ediBng
candidate)
Italy – Israel Actions

14. Other
NGS-­‐related
experiences
-­‐
Gene
expression
in
collaboraBon
with
Prof.
Giorgino
(UNIBA)
-­‐
Gene/exon
expression
in
renal
carcinoma
OMNIA
LH75
Automated
qPCR
for
validaBon
of
molecular
biomarkers

15. Acknowledgments
PRIN2009
PRIN2010
PRIN2013
MICROMAP
CNR-­‐Aging
program
http://www.arisla.org/
http://www.epigen.it/
Italy – Israel Actions