next generation sequencing applications
TRANSCRIPT
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
o Miller syndrome – USA - 2009 o TARP syndrome – USA - 2010 o Schinzel-Giedion syndrome -
Netherlands - 2010 o Fowler Syndrome – Canada - 2010 o Terminal Osseous Dysplasia –
Netherlands – 2010 o Hearing Loss – USA – 2010 o Perrault Syndrome – USA – 2010 o Kaposi sarcoma – USA – 2010 o Sensenbrenner Syndrome –
Netherlands – 2010 o Hyperphosphatasia syndrome –
Germany – 2010 o Kabuki syndrome - USA – 2010 o Van Den Ende-Gupta syndrome –
Canada – 2010
o Neonatal Diabetes Mellitus – France – 2010 o Autoimmune lymphoproliferative syndrome –
USA – 2010 o Familial Amyotrophic lateral sclerosis – USA –
2010 o Non-syndromic mental retardation – USA – 2010 o Osteogenesis Imperfecta –
Germany/Netherlands – 2011 o Hajdu-Cheney syndrome – London /France–
2011 o Acne Inversa – China – 2011 o Leucoencephalopathy – Japan – 2011 o Taybi-Linder syndrome – France – 2011 o Ochoa syndrome – Saudi Arabia – 2011 o Spastic paraparesis - USA – 2011 o Distal Arthrogryposis – USA – 2011 o 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
Erasmus Center for Biomics