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A Novel High throughput Custom Array based SNP Genotyping using Agilent platform Aiyaz Mohamed, Ajayakumar KM, Hemavathi K, Harsha OB, Saroja MK, Sudha Rao and Raja C Mugasimangalam *Presenting Author email: [email protected] I Genotypic Technology, Bangalore, India. www.genotypic.co.inDownload poster: http://www.genotypic.co.in/Domains/2/AgriGenomics.aspx
Microarray technology has been a reliable and effective tool for various genomics applications like Gene expression CGH and Location analysis. SNP arrays have been used widely in large-scale Genome-wide association studies in human and other species. However, the challenge of customization of arrays for large number of SNPs (10s of thousands to a Million) across moderate number of samples (from 10 to hundreds) remain unanswered. Genotypic Technology has developed a novel microarray based method - genotyping by hybridization using Agilent Sure print platform. We present here Array design, Experimental methods and Data Analysis. Work flow presented here provides immense opportunities for validation of data from large-scale NGS experiments and for low-cost medium throughput analysis of genome variants customizable for any content across any species. This will be most optimal and cost effective solution for sample sizes of few hundreds, and number of SNPs ranging from a few thousands to 50,000. Genotypic also offer a variety of custom microarray solutions, catalog array designs for plants and animal species, Haloplex and SureSelect designs as well full services for NGS and NGS analysis.
Abstract
Experimental Methodology
Analysis Workflow
Image Analysis by Agilent Feature Extraction Software
Microarray Raw Data (Signal intensity)
Background Subtracted signal intensities
SNP assay (4 sense and 4 antisense probes)
Base Calling by RightCallZygosity detection (Homozygous or Heterozygous)
Sense probe intensities
AntiSense probe intensities
Genome Sequence/Transcriptome sequence/NGS contigs/ SNP locus
Probe Design (RightDesign)
AMADID Generation
Plants/Cells/Tissues/Blood
Extract DNA
Quality Control ScanningLabel and hybridize to array
Analysis by RightCall Agilent Extraction software: Raw Data
Enlarged Image Microarray Image
T TA AG GC C
SNP calling by RightCall
Inte
nsity
Inte
nsity
Inte
nsity
Inte
nsity
Chr 1 SNP Call : A (Homozygous) Chr 2 SNP Call : C (Homozygous)Chr 2 SNP Call : C (Homozygous)
Chr 3 SNP Call : T/G (Hetrozygous) Chr 10 SNP Call : C/T (Hetrozygous)
This method and the work flow has been sucessfully applied in SNP genotyping of various species including Human and other mammalian species; Watermelon, Maize, Sorghum, Tomato and other plant species. This novel technology has practical applications in Plant breeding, Animal breeding and validation of NGS data. Ready to order Catalog SNP microarrays for Tomato and Human are available OR arrays can be customized for target SNPs and shipped to the user along with RightCall tool OR full services can be availed from Genotypic’s Agilent certified Lab.
Typically optimal assays can be made for 75% of the SNPs. Once optimized and filtered 95% of the assays are typically successful.
Example of a research publication where Agilent arrays are used to validate NGS resultsGenome Sequence Of The Pattern Forming Paenibacillus Vortex Bacterium Reveals Potential For Thriving In Complex Environments, Sirota-Madi A, Olender T, Helman Y, Ingham C, Brainis I, Roth D, Hagi E, Brodsky L, Leshkowitz D, Galatenko V, Nikolaev V, Mugasimangalam RC, Bransburg-Zabary S, Gutnick DL, Lancet D, Ben-Jacob E, BMC Genomics, 11, 710 (2010)
Conclusions
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RightDesign, developed by Genotypic was used for probe designing. Total of 8 Probes per SNP were designed with SNP as middle base (4 in Sense and 4 in Antisense strand). The length of the oligomer was optimized based on GC content and other criteria. These microarrays were then printed using Agilent SurePrint technology.
Array Design and Printing
Quality checked DNA samples were labeled using Agilent Sure Tag DNA labeling kit and were hybridized on the SNP microarray. The arrays were washed and scanned in the Agilent scanner as per Agilent array CGH protocol. Intensities of the probes were extracted using Agilent Feature extraction Software.
RightCall is a proprietary software tool developed by Genotypic was used to make SNP calls from the probe intensity data. Probe signal intensity values of sense and antisense strands were used for base call. The output is a user friendly excel table that can be imported into statistical analysis packages.
Microarray Experiment
SNP Microarray Screening: Data Analysis
Chromosome Position Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
chr1 98158 A T A A/T A/Tchr2 983674 G/A G/A G/A - G
chr5 143201 C G C/G C C/G
chr11 561796 A - T A A
chr12 644342 A A A A A
