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© 2015 - Diversity Arrays Technology Pty. Ltd KDDart - Knowledge Discovery System Andrzej Kilian Diversity Arrays Technology Pty Ltd CBA workshop Canberra December 4 2018
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
Andrzej KilianDiversity Arrays Technology Pty Ltd
CBA workshop CanberraDecember 4 2018
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ Introductions¨ What is important in good genotyping
¡ People ¡ QC/QA systems¡ Informatics¡ Equipment
¨ DArT PL’s genome profiling methods¡ Whole genome “random distribution” markers (DArTseq, DAtRseqLD)¡ Targetted genotyping (DArTag, DArTmp, DArTcap, rhAMPseq?)
¨ Examples of applications¨ Comprehensive support in data analytics and management¨ Concluding remarks
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ Mission: Actively support more effective and sustainable use of natural resources¨ Main business: genome profiling services and IT support for agriculture and ecology¨ Operating since 2001 – first affordable whole genome profiling service ¨ Global service provider: >1.5 million whole genome profiles, >1000 organisms¨ Supporting international agriculture through tech development, training & service¨ Collaboration in technology package transfer (Mexico, Africa) ¨ Team of 40 (lab, data analysis and IT) + international visitors/trainees ¨ High diversity of clients: From farmers to multinational corporations, majority from OS,
SMEs and public (CGIAR) most involved in breeding
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OUR PEOPLE
OUR CLIENTS
SAGA
IGSS
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ 37 years ago: PhD in pop genetics and microevolution with 8(!) isozymes (Arabidopsis thaliana)
¨ 33 years ago: RFLP in Cambridge UK
¨ 25 years ago: plant gene cloning in USA with RDA and RFLP subtraction –foray into genomic representations
¨ 21 years ago: invention of DArTmethod – combining RFLP concepts with genomic representations
¨ 10 years ago: DArTseq – initial transition from array platform to NGS platforms
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ Always start with learning about user needs!
¨ Price of service is only at the end of the “meeting of the minds” process
¨ Technology applied is often not what was initially requested ¡ Finding a better way to deliver required outcome
¡ Dependent on application, volume of service etc
¨ Complementing the “missing bits” in clients’ technological capacity¡ Many molecular marker options (from dozens of
markers/sample up to whole genome analysis)¡ Dedicated analytics/data mining integrated with marker data
production
¡ KDDart platform for data integration
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ Not only our own staff!
¨ Over 100 trainees since inception
¨ Predominantly short term visitors
¡ 2 weeks average stay
¡ Usually with materials for processing
¡ Good understanding of all processes ¡ Joint downstream data analysis
¨ Over 20 longer term several months +
¡ Mostly in the context of technology transfer
¡ Competence in performing the relevant tasks and troubleshooting
¡ Established technology in Mexico (partnership with CIMMYT) and now in Kenya (BMGF grant, partnership with ILRI) - IGSS
¡ IGSS operating using DArTseq platform, preparing to roll out other DArT methods and some of the IT tools
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ 18 years of continued development –nearly 100 releases
¨ Online Ordering and LIMS/database integration: critical for sample tracking:
¨ Integrated with: ¡ Raw (sequence) data production¡ Sequence curation, barcode splitting¡ Imaging systems¡ Pipetting robots, etc...¡ Secondary pipeline (DArTsoft14)
¨ Raw data stored from start of DArT operations
¨ Data complexity/volume reduction¨ Coping well with 2-4 fold yearly increase in
sample/data volumes
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ 20 years of technology development (complexity reduced representations)
¨ Complexity reduction optimised for each organism¡ Pair of Restriction Enzymes, at least one methylation
sensitive
¨ Targeting c 200,000 mostly low copy sequences (methyl filtration)¡ In most cases >90% of markers single copy
¨ Most assays read at >10X
¡ High call rate
¡ High data quality
¨ Extensive quality control including QC individual libraries
¨ Technical replication -> selection of best markers (SNPs & SilicoDArTs)
¨ Sequencing platform independent
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ SNPs and SilicoDArTs provide complementary picture of diversity
¨ SilicoDArT analysis provides additional QC insights for SNPs
¨ SilicoDArTs slightly better in technical reproducibility (higher read depth)
¨ Insight into epigenetic variation via SilicoDArTs
¨ Better performance in ”deep” phylogenetics compared to SNPs
Typical results:(average of 100 services using 2.5 million reads option)
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SNPs SilicoDArTs
Number 53,860 44,570
Read depth 25 29
Reproducibility 99.5 99.8
Call rate 89.7 96.2
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ DArTseqLDworks well when medium density of markers in random positions across the genome is required/sufficient. ¡ Representations around 10x smaller than in DArTseq. ¡ Method optimisation (rare cutting RE combinations) but no
specific oligo synthesis required for each organism. ¡ Offers a cheaper setup and no ascertainment bias when
applied to new material¡ Very simple analytics¡ Applied by many commercial and public GS programs and in
lower resolution diversity analysis (genebanks, ecology)
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
Targeted genotyping complements DArTseq
q Four complementary platforms developed for GS and other lower density applications
q Platform choice depends on required marker density and the volume of demand
q Sequencing platform for assay readout
q Adopted several methods for sequence capture
q Performance, cost and FTO considerations
q Average read depth/locus >100 – very high data quality
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DArTmpdeveloped recently for applications in which low density of markers is sufficient (<500). The method involves two step PCR process: 1. Selecting short stretch of sequence around the SNP 2. Barcoding and NGS platform elements addition
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ DArTcap: regular DArTseqassay followed by selected markers capture with “baits”
¨ Enriched library sequenced. ¨ Outperforms other low(er)plex
assays in polyploids thanks to complexity reduction step.
¨ Probe synthesis investment required and some ascertainment bias possible if probe set not optimised for material/application
¨ Enabling “exom/gene space capture” in the absence of reference sequence and/or transcriptome data (more effective in plants!)
Standard DArTseq library construction
All fragments have a complete assembly of elements required for Illumina sequencing
Hybridisation with biotin-RNA probes complementary to markers of interest
Fragments with markers of interest pulled down with magnetic beads
Captured fragments eluted and Illumina sequenced – DArTcap markers extracted
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ Comprehensive characterisation of germplasm/diversity studies: ¡ Parents of breeding programs¡ Germplasm collections (e.g.SeeD project)
¨ Genetic ID/PBR¨ Seed purity/product quality testing
¡ Partnership with GRDC to deliver testing for grains in Australia, also internationally (mainly Africa)
¨ Genetic and QTL mapping¡ > 1000 mapping populations
¨ Association mapping/GWAS ¡ “Cloning as you go” in breeding programs
¨ Gene tagging /cloning (under $10,000) - quantitative BSA
¨ Genomic Selection (plants and animals)
¨ Population genetics¨ Eco-evo, applied ecology ¨ SpIDer: sort of “barcode of life” (species identification)
¡ long term project with Chevron in Australia
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© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ Cost of assay and platform choice driven by size of demand and application type – consultation critical
¨ Limiting the number of platforms => lower investment (sequencing “the best bet”)
¨ Assay densities
¡ Hundreds of thousands for parents
¡ Thousands for selections/biparental crosses
¡ Dozens for “targeted selection”
¨ High density~$35-40/sample depending on volume
¨ Cheaper versions of assay ($10-25)
¡ With lower depth with imputation (“on the fly”)
¨ Markers from the same representation -> efficient imputation
¨ Low density (100- 1000 markers) – $7-10
¨ Lowest densities ~ 30 markers ($2-4)
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Breeding parents(>100,000 markers)
Routine(GS) selection(>5,000)
Targeted selection
(>30)
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
q Initial method described by Bedo et al, BMC Genetics (2008) – partnership with NICTA
q Applicable to any population type including breeding programs
q Used either for QTL/GWAS or for genomic selection
q GEMir and GEMisq New technology for finding interacting
pairs of genes (or higher level interactions)
q Adding interactions of markers with environment
q Testing capacity to improve prediction models of GS with “epistasis”
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GWAS analysis – linear models
GWIS analysis – interaction search
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System 20
Data production Data integration
© 2015 -Diversity Arrays Technology Pty. LtdKDDart -Knowledge Discovery System
¨ Integration with DNA profiling and IT services provided by DArT PL and IGSS ¡ Consultancy with users to find the best solution to their problem
¨ DArTseq a generic, scalable and robust platform for any organism and application (over 1,000 species in routine analysis)
¨ Support in lower density assay development and delivery¡ DArTmp, DArTag, DArTcap and DArTseqLD
¨ DArTdb and DArTsoft14 in support of marker data production
¨ KDDart platform for data storage and analysis
¨ Continued development of genotyping methods (DArTreseq, whole genome assembly/structural variants) and free tools for data collection and analysis
¨ Open access and partnership model for technology and service delivery
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