NGS data analysis and molecular breeding pipeline
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The document describes ISMU, a pipeline for NGS data analysis and facilitating molecular breeding. ISMU version 1 focuses on SNP discovery between genotypes through mapping, assembly, and visualization. Version 2 applies identified SNPs to breeding through assay design, genotype calling, and analysis. The pipeline benchmarks open source programs, performs assembly and polymorphism detection between genotypes, and identifies parental lines for molecular breeding applications. It provides user-friendly interfaces for uploading data and visualizing results. Future plans include updating tools, extending pipeline capabilities, and linking with other databases and analysis systems.
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NGS data analysis and molecular breeding pipeline
- 1. ISMU pipeline for NGS data analysis and facilitating molecular breeding http://hpc.icrisat.cgiar.org/NGS/
- 2. • Short read length of sequences • Availability of many tools • Platform dependency and command line driven • No direct ways for prediction of SNPs between genotypes • Quality scores vary depending on version and technology Challenges
- 3. ISMU version 1 • SNP discovery from NGS data – Pipeline for mapping / assembling – Calling SNPs between genotypes – Visualisation
- 4. ISMU version 2 • Application of identified SNPs to breeding
- 5. • Benchmark available open source short reads assembly and downstream analysis programs/software. • Assembly and polymorphism detection between genotypes and visualization • Assay design (Illumina GoldenGate Assay), genotype calling and visualization and analysis of SNP genotyping and haplotype data • Identify and use parental lines for using in MABC or MARS • Discovery of SNP markers for use in foreground and background selection of MABC or MARS. • Documentation of the pipeline and the integrated software. Objectives of NGS Pipeline
- 6. Control Flowchart ICRISAT CROPS YesNo Input Data & validation Upload Reference & data Mapping (Maq,Novo) Mapped reads Assembly Visualization Consensus calling Report SNPs • Extract sequences with SNPs • Design primers • In silico validation by SNP2CAPS Database ADT Score G.G Assay Bead Studio Flapjack
- 7. Genotype 1 Genotype 2 Chrom1 Pos RefAllele Gtyp1 Gtyp2 5 303 A G ? Maq NovoProgramme SNP Bet Genotypes Standard Methodology Mapping Mapping Assembly SNP Calling ag. Reference ADT Scoring Reporting Remove duplicates Check the inverse combination Compare allele between genotypes Base calling in 2nd genotype Predicted SNPs against Reference
- 8. Customized Methodology (Consensus Base Calling-cc) ccMaq ccNovo SNP Calling Genotype 1 Genotype 2 Programme Inhouse Script ADT scoring Genotype 2 fmaj=21/28 =0.75 Genotype 1 fmaj =38/40 =0.95 Mapping Mapping
- 9. Consensus Base Calling Parameters (Default) • Max number of mismatches <= 7 • Sum of mismatches score <=60 • Min mapping quality =>0 • Read depth threshold =>5 • Major base frequency threshold => 0.75
- 10. What if more than 2 genotypes? Genotype1 Genotype2 Genotype3 Genotype4 G1 G2 G3 G1 0 1 1 G2 0 0 1 G3 0 0 0 Combination of genotypes = (n2–n)/2
- 11. • Reads format fna and qual (Standard/Sanger)Fastq SCARF fomat Solexa fastq, Solexa export AB SOLiD read format FASTA • Reference sequence Chickpea transcript assembly Pearl millet transcript assembly Pigeonpea transcript assembly Medicago genome Sorghum genome NGS pipeline input data
- 12. NGS pipeline (Input 1) http://hpc.icrisat.cgiar.org/NGS/
- 13. NGS pipeline (Input 2)
- 14. NGS pipeline (Help page)
- 17. Available in 2 Editions 1. Server Edition 2. Desktop Edition Pipeline Editions
- 18. • User friendly web interface – Installation on following Linux platform • Fedora 13 • Cent OS 5 • Clients can be any OS with a web browser • Communication resources • SMTP (Email) • Session specific job processing - Avoid file over writing Server Edition
- 19. Desktop Edition • All functionalities of Server Edition on a Desktop • Supported OS • Fedora 13 • RHEL 5 • Single command installation • Available in Installable CD
- 20. Future plans •Consideration of new tools to integrate / update eg: BWA, Bowtie •Implementation of the extension to the pipeline •Evaluate cloud computing and high performance computing cluster options •Initiatives such as iPlant (discovery environment – genotype to phenotype)
- 21. • Identification of appropriate modules for MARS, GWS and GBS • Integration of MARS and GWS module • Linking of ISMU pipeline with DMS of IBP • Documentation & Training of ISMU pipeline Future Plans: ISMU v 2
- 22. Internet Architecture Reference Sequences Velvet Perl Prog Maq Novo CGI SNP Database Files downloading Dynamic Querying Assembly Visualization Input data validation NGS Data Analysis pipeline at ICRISAT Apache Server Hosting Web Pages SMTP Server
- 23. • Rajeev K. Varshney • Abhishek Rathore • Jayashree B • Vivek Thakur • R. Pradeep • A. Bhanu Prakash • Sarwar Azam • G.Meenakshi • David Marshall • Iain Milne Contributors • Jonathan Jones • David Studholme • Greg May • Andrew Farmer • Jimmy Woodward • Dave Edwards