NGS Illumina sequencing: a pratical overview - Centri di Ricerca ...
NGS Illumina sequencing: a pratical overview - Centri di Ricerca ...
NGS Illumina sequencing: a pratical overview - Centri di Ricerca ...
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Tecniche per la produzione <strong>di</strong> dati genomici<br />
<strong>NGS</strong> <strong>Illumina</strong> <strong>sequencing</strong>:<br />
a <strong>pratical</strong> <strong>overview</strong><br />
Federica Cattonaro<br />
Istituto <strong>di</strong> Genomica Applicata<br />
IGA Technology Services Srl<br />
(U<strong>di</strong>ne, Italy)<br />
Convegno ‘La genomica al servizio dell’agricoltura’<br />
Università Cattolica del Sacro Cuore<br />
Piacenza, 25 maggio 2012
<strong>Illumina</strong> target enrichment protocol
TARGET ENRICHMENT OPTIONS<br />
Custom target<br />
You can choose which region and for which organisms (need a reference) to do<br />
genome capture<br />
Region captured from 200Kb to 32Mbp for Agilent SureSelect system<br />
Region captured from 700Kb to 15Mbp for <strong>Illumina</strong> system<br />
Software to design the probes: <strong>Illumina</strong> Design Stu<strong>di</strong>o, Agilent eArray<br />
ONLY human target re<strong>sequencing</strong><br />
Human all exon<br />
Agilent SureSelect all exon 50Mbp= target size about 50 Mbp<br />
<strong>Illumina</strong> TrueSeq exon enrichment kit = target size about 62Mbp<br />
Custom target re<strong>sequencing</strong><br />
Amplicon Seq (<strong>Illumina</strong>): 12 to 96 Kbp<br />
Halopex (Agilent): until 1Mbp
SureSelect All Exon – Towards non-Human Species<br />
All Exon Mouse:<br />
49.6Mb design. Exon definition derived from Ensembl + RefSeq,<br />
designed against mm9 reference from UCSC<br />
All Exon Bovine:<br />
64Mb design. The library is based on University of Maryland build 3.1<br />
against NCBI exons as well as pre<strong>di</strong>cted exons, UTRs, and miRNAs.<br />
Worked with USDA on design.<br />
All Exon Canine:<br />
54Mb design. UCSC tracks from CanFam2 for ensembl as well as<br />
Refseq, human protein alignments, and spliced ESTs that lie outside of<br />
ensembl.<br />
All Exon Zebrafish:<br />
96Mb design. Zv9 library is in Emsembl and is annotated with gene and<br />
exon coor<strong>di</strong>nates. Worked with Sanger on design.<br />
Note: All Exon Bovine, Canine and Zebrafish are being provided in Early Access<br />
Format
1. <strong>Illumina</strong> TruSeq DNA Sample prep bisulfite mo<strong>di</strong>fied<br />
(C to U changes in non metylated cytosine)<br />
1. ME-DIP analysis (antibody vs methylated C)
Bioanalyzer, Qbit fluorimeter, Real-Time....
IGA Technology Services S.r.l.<br />
(commercial spin-off of IGA)<br />
<strong>NGS</strong> facilities<br />
<strong>Illumina</strong> Genome Analyzer IIx (April 2010): 95 Gbp/run (pair-end 2x150bp)<br />
(already <strong>di</strong>smissed!)<br />
HiSeq2000 (by December 2010): 600 Gbp/run (pair-end 2x100bp)<br />
MiSeq (by April 2012): 2 Gbp/run (pair-end 2x150bp)
Esempi <strong>di</strong> servizi alla ricerca legati al<br />
sequenziamento <strong>NGS</strong><br />
applicazione attività<br />
Metagenomica sequenziamento <strong>NGS</strong> <strong>di</strong> comunità ambientali (16S o tutte le<br />
molecoile <strong>di</strong> RNA/DNA)<br />
Analisi <strong>di</strong> espressione<br />
genica<br />
sequenziamento <strong>NGS</strong> dell’mRNA (trascrittoma) per l’analisi<br />
dell’espressione genica in specie vegetali, in bovini, cani, uomo...<br />
Analisi <strong>di</strong> smallRNA annotazione e quantificazione<br />
Ricostruzione<br />
trascrittomi<br />
Sequenziamento <strong>di</strong><br />
piccoli genomi<br />
Identificazione<br />
varianti SNP e INDELs<br />
sequenziamento <strong>NGS</strong> per la ricostruzione delle sequenze co<strong>di</strong>ficanti<br />
(geni) in varie specie il cui genoma non è stato sequenziato, come<br />
tasso, abete bianco, anemone <strong>di</strong> mare, crostacei...<br />
sequenziamento <strong>NGS</strong> <strong>di</strong> ceppi batterici<br />
Sequenziamento esomi umani, tumori (controllo vs tumore), target<br />
re<strong>sequencing</strong>
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