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Ngs and Bioinformatics FÉDÉRER LA RECHERCHE ET L’INNOVATION MÉDICALE EN CANCÉROLOGIE NGS AND BIOINFORMATICS Yannick BOURSIN Jean-Charles CADORET Marc DELOGER M’Boyba DIOP Pierre GESTRAUD www.canceropole-idf.fr Jean-François OUIMETTE NGS & Cancer : Analyses RNA-Seq Nicolas SERVANT 7, 8, 9 novembre 2018 2 Outline 1- INTRODUCTION a) Sequencing technologies b) Sequencing library technologies c) Bioinformatics part of sequencing projects d) The RNA world e) RNA-seq usage in oncology 2- QUALITY CONTROLS ON RAW READS a) Raw reads formats b) Quality controls 3- SHORT READS ALIGNMENT AND QUALITY CONTROL a) Reads alignment b) Alignment formats c) Quality controls on aligned data d) And then ? NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 A short introduction SEQUENCING TECHNOLOGIES NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 4 NGS technologies S. Baulande, I. Curie NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 5 Hiseq, Pacbio, PGM, … What does that mean ? Platform Provider Reads Max Reads Size Throughput Time Number (bp) (Gb) Novaseq Illumina 20000 M 2x150 6000 <3 days Hiseq X Illumina 6000 M 2x150 1800 <3 days Hiseq Illumina 5000 M 2x150 1500 <7 days NextSeq Illumina 400 M 2x150 120 <2days S5 Ion Torrent 80 M 200 15 2-4 hours MiSeq Illumina 25 M 2x300 15 40 hours MiniSeq Illumina 25 M 2x150 7.5 24 hours S5 Ion Torrent 12 M 600 4.5 2-4 hours PGM 318 Ion Torrent 4 M 400 >1 2-4 hours PGM 316 Ion Torrent 2 M 400 >0.1 2-4 hours PGM 314 Ion Torrent 0.5 M 400 >0.01 2-4 hours Sequel Pacific 385 K – 6M 20000 8 – 128 <6 hours Bioscience (1-16 cells) (1-16 cells) NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 6 NGS Market (2013) http://enseqlopedia.com/ngs-mapped/ S. Baulande, I. Curie NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 7 Pacific Biosciences (SMRT) Single molecule real time (SMRT) sequencing technology http://www.pacb.com/ NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 8 Pacific Biosciences (SMRT) Main Applications : • De Novo Assembly • Haplotype phasing • Isoform detection • DNA modifications • Repeated elements • … But : • High error rate (15%) • High cost (~ 1200 euros / smrtcell) • Low input http://www.pacb.com/ NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 9 Oxford Nanopore (MinIon) A nanopore is a nano-scale hole. In its devices, Oxford Nanopore passes an ionic current through nanopores and measures the changes in current as biological molecules pass through the nanopore or near it. The information about the change in current can be used to identify that molecule. Summer 2018 MinION specs : 512 nanopores per flowcell (~900 euros + 190 wash kit) >8 Gbase per run (48h movie ~ 10-20Gb) 1D² Sequencing Kit (R9.5 SQK-LSK308) = 450 bases per second (~600 euros) Read length ~ Fragment length MinKNOW software 2.0 NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 10 Oxford Nanopore (MinIon) Main Applications : • Direct DNA/RNA-seq • Clinical applications • Portability • Real-time data • Direct molecular analysis But : • High error rate (no consensus compensation) • High cost (~1600 euros per run) • Problems with homopolymers • Strong context effect for modified base detection A MinION Starter kit includes a library preparation kit which prepares purified genomic DNA, amplicons or cDNA ready for loading onto the MinION, in under two hours. https://nanoporetech.com/ NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 A short introduction SEQUENCING LIBRARY PROTOCOLS NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 12 Different libraries for different applications Single-end sequencing Length of end sequences: depends on the platform -> ChIP-seq (TFs), small RNA-seq, amplicon-seq, genome-wide CRISPR-Cas9 screens et RNA-seq (DGE) Paired-end sequencing Sequence both ends of DNA fragment Insert size: <800nt Length of end sequences: depends on the platform -> DNA-seq (WGS, WES, etc…), RNA-seq, ChIP (histone marks) etc... NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 13 Different libraries for different applications Mate pair sequencing Sequence both ends of DNA fragment Insert size: >500nt Length of end sequences: depends on the platform -> Detection of large structural variations Illumina SOLiD NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 A short introduction BIOINFORMATICS PART OF SEQUENCING PROJECTS NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 15 Sequencing costs evolution (2011 presage) 12 Sboner, Sboner, The al. A.et (2011). real of sequencing:cost higher than you think!Biology Genome NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 16 Real cost of a sequencing project (2016) Muir, P. P. Muir, et al.The (2016). real cost of sequencing: scaling to keepcomputation pace with data generation NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 17 Real cost of a sequencing project (2017) genome analysis guiding treatment of paof analysis guidinggenome treatment - 1.6% 4.87% 45.87 % 15.52 % 30.59 % Weymann, Weymann, D. et al. The (2017). costand costof trajectory whole NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 18 Real cost of a sequencing project (2018) 49% generation sequencing gene targeted panels + Bioinformatician - salary and disk storage (not taken into account in this paper) 22% Marino, P. et al. (2018). Cost of cancer diagnosisCost of cancer usingal. next (2018). et P. Marino, NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 19 What is a Bioinformatician ? A bioinformatician is someone in between the Biology and the Informatics, who should be able to discuss with biology and/or medical staff and to bring them a support to solve a question. Different fields of applications (BIOinformatician , bioINFORMATICIANS, bioSTATISTICIAN, … ?) • Data management • Development of applications and interfaces • Data analysis • Methodological developments NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 08th February 2016 Non-Coding Genome 2016 - N. Servant 20 Big science and data tsunami Illumina NovaSeq 6000 system • 20 billion reads every 2 days (6000 Gb 2x150bp) • ~ 2,4 TB of raw data • ~ 2,8 TB of aligned data • = 5,2 TB + at least two time this amount of disk space for data analysis • To process 400 RNA-seq samples (50M reads each) in less than 48h, we need 1600 procs / 12TB RAM NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 08th February 2016 21 Non-Coding Genome 2016 - N. Servant Big science and data tsunami Illumina NovaSeq 6000 system For one year at maximum capacity (146 runs) • ~ 350 TB of raw data • ~ 410 TB of aligned data • = 760 TB + at least two times this amount of disk space for data analysis • Need >2 PB of space disk per year of usage Strong computational skills are mandatory to manipulate and process sequencing data NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 22 After the sequencing … • First steps: – Save your data – Ask for wet lab details: protocols (including library/insert size), nucleic acid quantity, quality, … • Saving will avoid you losing data – Because you do not remember what it is – Because your data medium gets old NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 23 After sequencing: data analysis Main challenges : • The rapid evolution of the high-throughput technologies • The rapid evolution of the bioinformatics solutions • The rapid evolution of the biological/medical knowledge NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 A short introduction THE RNA WORLD NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 25 Impact of NGS in the biology Chromosome Conformation Capture Methylation 4C, 5C, Hi-C Bisulfite-seq MedIP-seq TFBS Histone Modification ChIP-seq ChIP-seq Nascent TRS Variants Detection GRO-seq DNA-seq Exome-seq Quantification of mRNAs and small RNAs RNA-seq Translational Alternative splice Efficiency variants Ribo-seq RNA-seq Alternative poly-A site PolyA-seq Adapted from Simon & Roychowdhury, Nature Reviews Drug Discovery (2013) NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 26 RNA-seq applications Total RNA sequencing lncRNA Low input sequencing PolyA + sequencing <10ng Gene expression Isoform detection Single cell RNA sequencing Fusion gene From 10pg RNA-seq Small RNA-sequencing miRNA and others smallRNAs Full Length RNA sequencing Long reads Others applications : PolyA- seq, CLIP-seq, CAGE-seq, Nascent RNA sequencing etc. NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 27 Two main types of library preparation S. Baulande, I. Curie NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 28 Technical variation in RNA-seq Main sources of technical variation : • Take tissue sample from individual • RNA extraction • cDNA conversion • Sequencing (PCR, etc.) • Day, laboratory, experimenter, machine • etc. NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 29 RNA vs small-RNA sequencing mRNA piRNA rasiRNA siRNA Coding RNAi miRNA Non RNA Coding lincRNA antisens tRNA rRNA snRNA Adapted from Weihua Zeng & Ali Mortazavi. 2012 FORMATION “NGS & CANCER : ANALYSE DE DONNEES RNA-SEQ” 12 - 14 NOVEMBRE 2014 NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 30 RNA-seq: applications RNA-seq small RNA-seq • Abundance of mRNA and differential • Abundance of ncRNAs and differential expression at the gene/transcripts level expression • Detection of transcripts • Enrichment of ncRNAs • Detection of fusion genes • Profiling of ncRNAs families • Variant calling: • Annotation of known ncRNAs • RNA editing • De novo prediction of miRNAs • Monoallelic expression • Detection of piRNAs • Viruses/Phages/Bacteria expression • … • … NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 31 (s)RNA-seq: bioinformatics tips RNA-seq small RNA-seq • Depending on the question: • 50bp single reads, Illumina Gene-scaled analysis: - 100bp single-end reads, Illumina • 20-40M reads - 30M reads Fusion genes detection & Transcript-scaled analysis: - 100bp paired-end reads, Illumina - 75M pairs Rare events: 100M pairs At least 3 biological replicates for group comparison NGS & Cancer : Analyses RNA-Seq 7, 8, 9 novembre 2018 32 RNA stranded sequencing Most of the RNA-seq protocols are now directional.
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