The SMRTer Way: Single Genes to Complex Genomes

Transcription

1 The SMRTer Way: Single Genes to Complex Genomes Ulf Gyllensten, Professor Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden

2 Topics National Genomics Infrastructure (NGI). PacBio from single genes to complex genomes.

3 National Genomics Infrastructure (NGI) Among the five largest European sequencing centers. Core facility open to Swedish research groups. MPS sequencing, Sanger sequencing and genotyping. Funded as a National Research Infrastructure by SciLifeLab, Swedish Research Council (VR-RFI) and KAW Foundation.

4 MPS technologies at NGI Short-read MPS Long-read MPS

5 Analysis cluster and storage of MPS data From reads to. assembled genomes ~3 M cpuh/month on a dedicated cluster ~7 PB storage. Long-term storage in archive. CPU with extra large memory (2TB)

6 PacBio sequencing at NGI/Uppsala Two Pacific Biosciences RSII systems June 2013 August 2014

7 PacBio Data production in Uppsala

8 Assembly projects BACs, YACs, fosmids, plasmids, Gram positive and negativembacteria Archaea Parasitic protists Fungi (yeasts, mushrooms) Algae Mosses Higher plants Worms Butterflies, Insects Birds Lizards Fish Mammals

9 Applications on PacBio Non-clinical applications Complete genomes BACs/YACs/plasmids 16S rrna Gap filling Whole transcriptome sequencing Isoform discovery Amplicon sequencing Mutation detection Haplotype phasing Target re-sequencing Metagenomics Procaryotic methylation Clinical applications Chronic Myeloid Leukemia Acute Myeloid Leukemia HLA sequencing Repeat expansions Infection screening

10 PacBio applications A. Small genome assembly B. De novo complex genome assembly C. Targeted sequencing

11 Small genome assembly - PacBio the method of choice for small genomes. - Sample quality is crucial. Good quality an (almost) complete genome, poor quality partial or no genome. Example:

12 Complex genomes: De Novo Assembly of Rabbit Genome Two for one genome : Assembly of an F1-hybrid between two subspecies of rabbit. PI: Professor Leif Andersson, Uppsala Aims: Create a New reference assembl/y/ies In depth characterization of loci exhibiting strong allele frequency shifts around hybrid zone between O. c. coniculus and O. c. algirus in Spain. 2 % of genome shows dramatic reduction in ability to spread to other side, rest of genome leaks into other side.

13 The order Lagomorpha consists of two families: Leporidae (hares and rabbits) and the Ochotonidae (pikas) Likely radiated from common ancestor in Asia 60 million years ago European rabbit (Oryctulagus coniculus) and the closest extant species, the hispid hare (Caprolagus hispidus)in South Asia diverged approximately 7-10 million years ago, like most of the Leporidae Lagomorpha Evolutionary History of Lagomorphs in Response to Global Environmental Change, PLoS One, April 2013 Volume 8 Issue 4

14 Origin and domestication of the European rabbit (O. cuniculus) O. c. coniculus Dispersal to southern France O. c. algirus

15 Strategy and challenges 300 SMRT cells (around 200Gb) run in Uppsala O.c.c x O.c.a hybrid 6 BioNano runs (by BioNano) Parents of F1-hybrid sequenced to 30x using PCR free Illumina libraries. BAC-ends and phosmids from Sanger assembly (250k & 2 million respectively) Sanger assembly OryCun2 (2.74 Gb) Falcon diploid assembly attempted Very high heterozygosity!

16 De Novo Assembly of Rabbit using BioNano 6 runs conducted with 400 Gb of molecules >150kb Raw Data (molecules > 150 kb) Initial Assembly High Depth Assembly Data input 184 Gb 367 Gb 367 Gb Stringent Assembly Number of genome maps Assembly size 2.57 Gb 3.76 Gb 4.44 Gb Genome map N Mb 1.4 Mb 1.07 Mb Longest genome map 4.5 Mb 6.4 Mb 6.3 Mb

17 Heterozygous Genome Maps are Produced Ref GM

18 SciLifeLab Whole Human Genome Initiative - WGS of patient cohorts (n=10,000 ind /year). - Establish a Genetic Variant Database for the Swedish Population (n = 1,000).

19 Population genomics projects The 1000 Genomes Project - genomes of 2500 unidentified people from 25 populations Genomics England: 100,000 whole genomes from patients by 2017.

20 The Swedish Genetic Variant Project A. Identify a cohort that reflects the genetic structure of the Swedish population. B. Generate WGS data using short- and long-read MPS technologies. C. Establish a user-friendly database to make information available to the research community (association analyses) and clinical genetics laboratories.

21 The Swedish Twin registry Inclusion based on twinning and distribution like population density. General population-prevalence of any disease. 10,000 individuals have been analysed with SNP arrays. Identify 1,000 individuals based on genetic structure and diversity across Sweden.

22 Principal components of European samples from 1,000 genomes project and 10,000 Swedish samples Finland Northern Sweden Southern- Central Sweden England and Scotland Italy Spain

23 European Individuals samples selected for from WGS 1,000 and 1000 genomes G EUR project and 1,000 selected Swedish samples Main genetic differentiation between Southern - Central and Sweden Northern Northern Sweden Southern Central Sweden

24 Step 1: WGS of Swedish control cohort Short-read Illumina X-Ten data to 30X coverage of the 1,000 individuals. Standard pipeline (GATK) for variant calling (SNP and indels). Construct user-friendly database for the community to make use of the data. Status: Identification of a control cohort Q Short-read MPS Q Data base implementation Q

25 Database for genetic variants CanvasDB (CANdidate Variant Analysis System and Data Base) Stores genetic variants with annotations, such as prediction of the functional consequence. At present the 3.1 billion genetic variants in the 1000 Genomes project. Search time not proportional to database size. Filter tools for analyses of monogenic and complex genetic disease analyses.

26 The Present Human Reference is Not Complete Some regions have been recalcitrant to closure with short-read MPS technology. Structural variation makes it difficult to assemble a truly representative genome. Long-read whole human genome sequencing provide the information.

27 Genome reference standards Platinum genome sequence A contiguous, haplotype resolved representation of the entire genome. Gold genome sequence A high-quality, highly contiguous representation. Silver genome sequence Standards TBD. Non-trio, PB/BN, no Bac library.

28 Gold Genome Sequencing Approach

29 The Human Reference Genomes Project Gold Reference Genomes Platinum Reference Genomes HG00733 HG00514 NA12878 CHM1 CHM13 NA19240 NA19434

30 New Reference Human Genome Sequences Platinum Genomes CHM1 An integrated assembly of Illumina, PacBio, BAC and BioNano data. CHM13 PacBio data assembly + BioNano data. Gold Genomes NA19240 Yoruba trio child; assembly completed. HG Puerto Rican trio child; sequencing in progress. HG00514 Han Chinese trio child, Q NA19434 Luhya (Kenya) trio child, Q

31 Step 2: WGS of Swedish cohort Establish Swedish reference genome sequences by de novo assembly of long-read Pacific Biosciences data (+BN). Ref genome individuals

32 First Swedish PacBio WGS First PacBio Assembly 20 kb library 157 SMRT cells 140 Gb data (~45X) FALCON assembly # of contigs (>=0 bp) 7708 # of contigs (>=1000 bp) 7653 Total length (>=0 bp) 2844 Mb Total length (>=1000 bp) 2844 Mb No of contigs 7692 Largest contig Total contig length N50 N Mb 2844 Mb 4.35 Mb 1.97 Mb

33 Step 3: WGS of Swedish control cohort Targeted long-read sequencing of regions of high medical importance (HLA, Trinucleotide expansion repeats). Resolve structural variation and repeats. Phase variation in repetitive regions and individual alleles. Study the methylation pattern in native DNA.

34 Methods for Targeted PacBio sequencing Long-range PCR. Target enrichment by hybridisation using DNA or RNA probe arrays. Amplification-free targeted enrichment.

35 Long-range PCR: HLA sequencing

36 HLA sequencing workflow 1. LR-PCR Amplification 4. PB Long Amplicon Analysis 2. SMRTbell prep 3. SMRT Sequencing 5. Allele identification (GenDx)

37 Long-range PCR: FADS FADS region has been under selection in human evolution Regulates the production of Omega-3/6 fatty acids (PUFA) Region is associated to many traits and diseases Two main haplotypes in humans: Ancestral and Derived

38 FADS project - functional variant at rs Functional variant for FADS1 expression identified! But is it linked to the Ancestral or Derived haplotype? Pan et al (submitted)

39 PacBio sequencing of FADS region Hybridization capture and pooled sequencing of FADS region: rs Results: AluYe5 rs > 1.2 kb rs Derived haplotype increases FADS1 activity Ancestral haplotype, reduces FADS1 activity

40 Targeted enrichment using DNA probe arrays

41 Targeted enrichment using RNA probes Modified version of PacBio+Agilent protocol

42 Capture of a ~2 kb library Reads mapped back to human genome

43 Off-target capture of gene not in probe design region MIC-B gene is captured because of high similarity to MIC-A! MIC-B MIC_A

44 De novo assembly of captured region A method to resolve structural variations and repeats Repeat length in example: bp Difficult or impossible to resolve with short reads

45 Amplification-free targeted enrichment Using Cas9 for targeting. Sequence native DNA. Compatible with multiple targets: HTT, FMR1, ALS & SCA10 in one reaction. Under development Input DNA SMRTbell library CAS9 targeting Sequencing

46 Technology Waves in Human Genome Analysis Genome Wide Association Studies Exome (Re-) Sequencing Short-read Genome (Re-) Sequencing Comprehensive Short-read Genome (Re-) Sequencing Whole-Genome De Novo Sequencing using long-reads. Jim Lupski: The Goal Is De Novo Assembly in the Clinic

47 What we sequence at NGI /

48 Who does the sequencing? Ulf Gyllensten Platform director Inger Jonasson Facility manager Olga Vinnere Pettersson Project coordinator Adam Ameur Bioinformatician, NGS Ignas Bunikis Bioinformatician, NGS Christian Tellgren-Roth Bioinformatician, NGS Susana Häggqvist Research engineer NGS Ida Höijer Research engineer NGS Cecilia Lindau Research engineer NGS Maria Schenström Research engineer NGS Magdalena Andersson Research engineer NGS Ulrika Broström Research engineer NGS Nina Williams Research engineer NGS Carolina Ilbäck Research engineer NGS Anna Petri Research engineer Sequencing Service Anne-Christine Lindström Research engineer Sequencing Service

49 What we sequence at NGI / THANK YOU