Center for Biomics Next Generation Sequencing Applications Wilfred van IJcken Erasmus MC Center for Biomics Biomedical Research Techniques (XVIth ed.), Nov 6
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 Peak detection Binding motifs Gata1 Gata1 only peaks Ldb1 Transcription Factors Tal1 Ldb1/Gata1/Tal1/ Eto2/Mtgr1 peaks Eto2 p300 Chromatin modifiers LSD1 Structural proteins CTCF Soler, van IJcken et al , Genes and Dev. 2010 Soler, van IJcken et al , Methods 2010
3C-Seq Detect DNA-DNA interactions close in 3D Stadhouders, ..,van IJcken et al .
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 Structural variants (SVs), Short InDels SNPs [e.g. kb-Mb-sized deletions, insertions, inversions, fusion genes] GATTTAGATCGCGATAGAG GATT------------GAG GATTTAGATCTCGATAGAG GATTTAGATCTCGATAGAG More difficult to detect than SNPs ~0.1% of the genomes of any presumably >0.1% of the genome two individuals differ due to SNPs
Example: Targetted sequencing H ypertrophic C ardio M yopathy prevalence 1 : 500 Main cause of sudden cardiac dead 50 gene panel Mybpc3, myh7 etc… 10 patients (multiplexed) indexing Bait design; Agilent Sureselect; ~50 genes, ~800 exons 1 MiSeq run PE 150 bp Alignment, Variant calling Clinical report Validation sanger sequencing of 10 mutations including indel
Targeted panel results Cardiomyopathy Old New Patient benefits Sanger Sequencing NGS 1. Diagnostic yield ↑ 33% 2. Turn around time ↓ 6 to 2 months 3. New type variants detected (indels + somatic mosaicisms) 2 genes 48 genes
Exome sequencing Exome = all coding regions (~ exons) of genome
> 200 Disease genes uncovered by Exome sequencing Neonatal Diabetes Mellitus – France – 2010 Miller syndrome – USA - 2009 o o Autoimmune lymphoproliferative syndrome – TARP syndrome – USA - 2010 o o USA – 2010 Schinzel-Giedion syndrome - o Familial Amyotrophic lateral sclerosis – USA – Netherlands - 2010 o 2010 Fowler Syndrome – Canada - 2010 o Non-syndromic mental retardation – USA – 2010 Terminal Osseous Dysplasia – o o Osteogenesis Imperfecta – Netherlands – 2010 o Germany/Netherlands – 2011 Hearing Loss – USA – 2010 o Hajdu-Cheney syndrome – London /France– Perrault Syndrome – USA – 2010 o o 2011 Kaposi sarcoma – USA – 2010 o Acne Inversa – China – 2011 Sensenbrenner Syndrome – o o Leucoencephalopathy – Japan – 2011 Netherlands – 2010 o Taybi-Linder syndrome – France – 2011 Hyperphosphatasia syndrome – o o Ochoa syndrome – Saudi Arabia – 2011 Germany – 2010 o Spastic paraparesis - USA – 2011 Kabuki syndrome - USA – 2010 o o Distal Arthrogryposis – USA – 2011 Van Den Ende-Gupta syndrome – o o Amelogenesis Imperfecta – UK - 2011 Canada – 2010 o
Whole genome sequencing Human and other species 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 Identify variation between individuals
Whole genome sequencing X Ten Outsource ? $1000 genome $1000 genome 30x 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) 10 weeks pregnancy 5% fetal DNA DNA isolation Prepare NGS Analysis Trisomy Report
NIPT: determine fetal chromosomal copy number Fetal cfDNA Maternal cfDNA Chr 21 Chr 21 Euploid Fetal Pregnancy Trisomy
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 me Doctor here is my genome and variation we need cheaper and faster High throughput sequencing we need educated doctors / clinicians
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
Erasmus Center for Biomics Genomics core facility at ErasmusMC www.biomics.nl w.vanijcken@erasmusmc.nl LNA
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