Experimental Designs in Next Generation Sequencing
- 1. Presented by Aishwarya Mohorikar Pawan Gutti Msc -1 Biochemistry The Institute of Science Mumbai Presented by Presented by Aishwarya Mohorikar pavan Gutti Msc -1 Biochemistry The Institute of Science Mumbai 4/4/2018 1 Experimental Designs in Next Generation Sequencing
- 2. • Introduction • Types of experimental designs • Basic NGS chemistry • Tools used in NGS • Good and Bad experimental designs 4/4/2018 2 Experimental Designs in Next Generation Sequencing
- 3. • Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionized genomic research. 4/4/2018 3 Experimental Designs in Next Generation Sequencing
- 4. • There are 3 types of experimental designs in NGS. • Paired v/s single • Multiple • Mate 4/4/2018 4 Experimental Designs in Next Generation Sequencing
- 5. Principle The Basics of NGS Chemistry In principle, the concept behind NGS technology is similar to CE sequencing. DNApolymerase catalyzes the incorporation of fluorescently labeled deoxy ribonucleotide triphosphates (dNTPs) in to a DNA template strand during sequential cycles of DNA synthesis. 4/4/2018 5 Experimental Designs in Next Generation Sequencing
- 6. Contd Library Preparation The sequencing library is prepared by random fragmentation of the DNA or Cdna sample, followed by5′and 3′adapterligation. Cluster Generation For cluster generation, the library is loaded in to a flow cell where fragment are captured on a lawn of surface-bound oligoscomplementary to the library adapters. 4/4/2018 6 Experimental Designs in Next Generation Sequencing
- 7. Contd Sequencing Illumina SBS technology uses a proprietary reversible terminator–based method that detects single bases as they are incorporated in to DNA template strands. DataAnalysis During data analysis and alignment, the newly identified sequence read are aligned to a reference genome . Following alignment, many variation s of analysis are possible, such as single nucleotide polymorphism (SNP)or insertion- deletion(indel) identification, read counting for RNA methods, phylogenetic or meta genomic analysis, and more. 4/4/2018 7 Experimental Designs in Next Generation Sequencing
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- 9. • Integrated Genome Browser Integrated Genome Browser (IGB) is an open source genome browser, a visualization tool used to observe biologically-interesting patterns in genomic data sets, including sequence data, gene models, alignments, and data from DNA microarrays. IGB reads data in dozens of formats, including BAM, BED, Affymetrix CHP, FASTA, GFF, GTF, PSL, SGR, and WIG. 4/4/2018 9 Experimental Designs in Next Generation Sequencing
- 10. Contd • Galaxy Galaxy was originally written for biological data analysis, particularly genomics. The set of available tools has been greatly expanded over the years and Galaxy is now also used for geneexpression, genomeassembly, proteomics, e pigenomics, transcriptomics and host of other disciplines in the life sciences. https://usegalaxy.org is the URL. 4/4/2018 10 Experimental Designs in Next Generation Sequencing
- 11. • the better approach for the above example is “not to” sequence all samples from an experimental group in a single lane, but make sure each lane contains samples from both the control and experimental groups. • That is where randomization comes in. One good NGS design is to randomly • pick three samples from control and experimental groups and sequence them in a lane. And sequencing the remaining six samples in the second lane. In this design even if one lane goes rogue that affects both the control and • experimental group samples equally and we still have one more “well • behaved” lane with both the groups. In statistical parlance, now the lane effect • and the experimental group effect is no longer confounded. 4/4/2018 11 Experimental Designs in Next Generation Sequencing
- 12. • A naive design is to put all six samples from the same group in a single lane (or dosequencing in one day). For example, multiplex all six control samples and sequence • them in one lane and multiplex all six experimental group samples and sequence in • the other lane. This is a bad design despite the fact that there are six biological • replicates and it is multiplexed. The reason why it is bad design is simply the same as the good old saying, “Don’t put 4/4/2018 12 Experimental Designs in Next Generation Sequencing
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- 14. A survey of tools for variant analysis of next- generation genome sequencing data BRIEFINGS IN BIOINFORMATICS. VOL 15. NO 2. 256 ^278 doi:10.1093/bib/bbs086 Advance Access published on 21 January 2013 Next-generation sequencing data interpretation: enhancing reproducibility and accessibility NGS analyses by visualization with Trackster Published in final edited form as: Nat Biotechnol. 2012 November ; 30(11): 1036–1039. doi:10.1038/nbt.2404. NIH Public Access 4/4/2018 Experimental Designs in Next Generation Sequencing 14
- 15. Thank you….. 4/4/2018 15 Experimental Designs in Next Generation Sequencing