Tecniche per la produzione di dati genomici

NGS Illumina sequencing:

a pratical overview

Federica Cattonaro

Istituto di Genomica Applicata

IGA Technology Services Srl

(Udine, Italy)

Convegno ‘La genomica al servizio dell’agricoltura’ Università Cattolica del Sacro Cuore

Piacenza, 25 maggio 2012

Illumina target enrichment protocol

TARGET ENRICHMENT OPTIONS

Custom target You can choose which region and for which organisms (need a reference) to do genome capture Region captured from 200Kb to 32Mbp for Agilent SureSelect system Region captured from 700Kb to 15Mbp for Illumina system Software to design the probes: Illumina Design Studio, Agilent eArray

ONLY human target resequencing Human all exon Agilent SureSelect all exon 50Mbp= target size about 50 Mbp Illumina TrueSeq exon enrichment kit = target size about 62Mbp Custom target resequencing Amplicon Seq (Illumina): 12 to 96 Kbp Halopex (Agilent): until 1Mbp

SureSelect All Exon – Towards non-Human Species

All Exon Bovine: 64Mb design. The library is based on University of Maryland build 3.1

against NCBI exons as well as predicted exons, UTRs, and miRNAs.

Worked with USDA on design.

All Exon Canine: 54Mb design. UCSC tracks from CanFam2 for ensembl as well as

Refseq, human protein alignments, and spliced ESTs that lie outside of

ensembl.

All Exon Zebrafish: 96Mb design. Zv9 library is in Emsembl and is annotated with gene and

exon coordinates. Worked with Sanger on design.

All Exon Mouse: 49.6Mb design. Exon definition derived from Ensembl + RefSeq,

designed against mm9 reference from UCSC

Note: All Exon Bovine, Canine and Zebrafish are being provided in Early Access

Format

1. Illumina TruSeq DNA Sample prep bisulfite modified (C to U changes in non metylated cytosine) 1. ME-DIP analysis (antibody vs methylated C)

Bioanalyzer, Qbit fluorimeter, Real-Time......

IGA Technology Services S.r.l. (commercial spin-off of IGA)

NGS facilities

Illumina Genome Analyzer IIx (April 2010): 95 Gbp/run (pair-end 2x150bp)

(already dismissed!)

HiSeq2000 (by December 2010): 600 Gbp/run (pair-end 2x100bp)

MiSeq (by April 2012): 2 Gbp/run (pair-end 2x150bp)

Esempi di servizi alla ricerca legati al sequenziamento NGS

applicazione attività

Metagenomica sequenziamento NGS di comunità ambientali (16S o tutte le molecoile di RNA/DNA)

Analisi di espressione genica

sequenziamento NGS dell’mRNA (trascrittoma) per l’analisi dell’espressione genica in specie vegetali, in bovini, cani, uomo...

Analisi di smallRNA annotazione e quantificazione

Ricostruzione trascrittomi

sequenziamento NGS per la ricostruzione delle sequenze codificanti (geni) in varie specie il cui genoma non è stato sequenziato, come tasso, abete bianco, anemone di mare, crostacei...

Sequenziamento di piccoli genomi

sequenziamento NGS di ceppi batterici

Identificazione varianti SNP e INDELs

Sequenziamento esomi umani, tumori (controllo vs tumore), target resequencing