Next Generation Sequencing Applications Wilfred van IJcken Erasmus - - PowerPoint PPT Presentation

Next Generation Sequencing Applications

Wilfred van IJcken Erasmus MC Center for Biomics Biomedical Research Techniques (XVIth ed.), Nov 6

Center for Biomics

Learning objectives

Previous presentation NGS: The basics  Background  Illumina sequencing technology  Terminology This presentation  Research applications  Diagnostic applications  Target enrichment  Future directions

Overview Sequencing Preparation Methods

RNA transcription RNA structure RNA low level DNA low level DNA rearrangements Methylation DNA-protein interactions

http://res.illumina.com/documents/applications/sequencing-technology-poster.pdf

NGS Applications

Gene expression analysis Discovery of novel transcripts, splice variants, miRNAs Protein-DNA/RNA interactions (ChIPSeq) genomic DNA interactions (3C, 4C, 5C Seq) Epigenetic profiling (DNA methylation) Targeted DNA sequencing Exome Sequencing Whole genome re-sequencing and De novo

ChIP-Seq

Detect protein-DNA interactions:

Chip-Seq

Gata1 only peaks Ldb1/Gata1/Tal1/ Eto2/Mtgr1 peaks

Soler, van IJcken et al, Genes and Dev. 2010 Soler, van IJcken et al, Methods 2010

Gata1 Ldb1 Tal1 Eto2 p300 LSD1 CTCF Transcription Factors Chromatin modifiers Structural proteins

Peak detection Binding motifs

3C-Seq

Stadhouders, ..,van IJcken et al.

Detect DNA-DNA interactions close in 3D

Epigenetic profiling (methylation)

Cytosine methylation (5-mC) has significant effect on gene expression and chromatin remodeling

Techniques: WGBS-Seq C to U with sodium bisulphite 5-mC stays C RRBS-Seq MspI digestion MeDIP Anti-5-mC antibody MIRA Capture with GST labeled protein Or 450k array

e.g. Carvalho, van IJcken et al. Epigenetics and chromatin 2012 doi:10.1186/1756-8935-5-9

Targetted sequencing

Custom or predefined probes

How does targetted sequencing result look?

Zoom in sequence result

Variation is not only SNP

GATTTAGATCGCGATAGAG GATTTAGATCTCGATAGAG ~0.1% of the genomes of any two individuals differ due to SNPs

Structural variants (SVs),

[e.g. kb-Mb-sized deletions, insertions, inversions, fusion genes]

presumably >0.1% of the genome GATT------------GAG GATTTAGATCTCGATAGAG

Short InDels

More difficult to detect than SNPs

SNPs

Example: Targetted sequencing

• Hypertrophic Cardio Myopathy
• prevalence 1 : 500
• Main cause of sudden cardiac dead
• 50 gene panel
• Mybpc3, myh7 etc…
• 10 patients (multiplexed)
• indexing
• 1 MiSeq run PE 150 bp
• Alignment, Variant calling
• Clinical report
• Validation sanger sequencing of 10 mutations including indel

Bait design; Agilent Sureselect; ~50 genes, ~800 exons

Targeted panel results

Cardiomyopathy

Old Sanger Sequencing 2 genes New NGS 48 genes Patient benefits

• 1. Diagnostic yield ↑ 33%
• 2. Turn around time ↓ 6 to 2 months
• 3. New type variants detected

(indels + somatic mosaicisms)

Exome sequencing

Exome = all coding regions (~ exons) of genome

> 200 Disease genes uncovered by Exome sequencing

• Miller syndrome – USA - 2009
• TARP syndrome – USA - 2010
• Schinzel-Giedion syndrome -

Netherlands - 2010

• Fowler Syndrome – Canada - 2010
• Terminal Osseous Dysplasia –

Netherlands – 2010

• Hearing Loss – USA – 2010
• Perrault Syndrome – USA – 2010
• Kaposi sarcoma – USA – 2010
• Sensenbrenner Syndrome –

Netherlands – 2010

• Hyperphosphatasia syndrome –

Germany – 2010

• Kabuki syndrome - USA – 2010
• Van Den Ende-Gupta syndrome –

Canada – 2010

• Neonatal Diabetes Mellitus – France – 2010
• Autoimmune lymphoproliferative syndrome –

USA – 2010

• Familial Amyotrophic lateral sclerosis – USA –

2010

• Non-syndromic mental retardation – USA – 2010
• Osteogenesis Imperfecta –

Germany/Netherlands – 2011

• Hajdu-Cheney syndrome – London /France–

2011

• Acne Inversa – China – 2011
• Leucoencephalopathy – Japan – 2011
• Taybi-Linder syndrome – France – 2011
• Ochoa syndrome – Saudi Arabia – 2011
• Spastic paraparesis - USA – 2011
• Distal Arthrogryposis – USA – 2011
• Amelogenesis Imperfecta – UK - 2011

Whole genome sequencing

Genome sequenced 2007 Craig Venter 7,5x 2008 James Watson 7,4x 2008 Han Chinese 36x 2008 Yorubian (nigeria) 30x 2008 Leukemia patient T/N 33x (14x) 2009 Seong Kim (korean) 29x 2014 100k genomes projects Human and other species Identify variation between individuals

Whole genome sequencing

X Ten $1000 genome 30x Outsource $1000 genome 40x

?

Human and disease, what to sequence?

• Most mendelian diseases are caused by exome mutations
• Exome is only ~1.6 % of human genome (50Mbp)

Panel Exome Whole genome

Genome >0,01% 1,6 % 95 % Sequencing 1/400x 1x 60x Interpretation ++ + + / - Validation ++ + + / - Speed ++ +

• Cost (est.)

€ 500 € 1000 € 5000

Comparision of exome and genome sequencing

Non invasive trisomy testing (NIPT)

DNA isolation Prepare NGS Analysis Trisomy Report

10 weeks pregnancy 5% fetal DNA

NIPT: determine fetal chromosomal copy number

Fetal cfDNA Maternal cfDNA

Fetal Trisomy Euploid Pregnancy Chr 21 Chr 21

NIPT in the news

Diagnostic applications

• Targetted sequencing

Cardio Myopathies, Ciliopathies, Cancer hotspot panel, Noonan, Neurodegenerative diseases, …

• Exome sequencing

Unknown disease, de novo

• Whole genome sequencing

Unknown disease, non-exonic

• Non invasive diagnostics

prenatal plasma T21 testing

• Cancer sequencing

germline mutations, therapy

• HLA typing

transplantation

Personalized medicine

• we need cheaper and faster High throughput sequencing
• we need educated doctors / clinicians

me

Doctor here is my genome and variation

Future

• Technical challenges resolved
• Implementation in tumor sequencing (free circulating tumor DNA)
• Inplementation in new born screening
• Replaces (partly) Sanger sequencing
• Interpretation challenge
• Ethical, legal, social issues
• Education doctors / clinicians
• Choice between panel, exome, whole genome
• Faster diagnosis (1 week from sample to diagnosis)

MinION

• USB sized sequencer
• One time use
• $ 900 dollar
• 500 nanopores
• >100 Mbp / h
• User defined runtime
• Lifetime electrodes is limiting

(days)

No sample prep Measure directly from blood

LNA Genomics core facility at ErasmusMC www.biomics.nl w.vanijcken@erasmusmc.nl

Erasmus Center for Biomics