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  1. 1. Presented by: Babu Lal Saini Ph.D. NGS and Its Application In Livestock ANIMAL GENETICS DIVISION ICAR-INDIAN VETERINARY RESEARCH INSTITUTE
  2. 2. Outline • Timeline of NGS platform • Introduction • First generation sequencing • NGS work flow • NGS Platforms • Application of NGS • Future challenges • Conclusion
  3. 3. Timeline of DNA sequencing technologies and platforms
  4. 4. Introduction “Knowledge of sequences could contribute much to our understanding of living matter”- Sanger DNA sequencing -process of determining the precise order of nucleotides Next-generation sequencing – High-throughput approach – Sequence several millions to billions DNA fragments in massively parallel manner nature.com/subjects/ngs Why NGS ???? – Sample preparation - faster and straightforward compared to sanger sequencing – Capability to generate massive volume of data – Accurate, fast and inexpensive method – Overcomes the limitation of microarrays to detect poorly expressed genes – More than a billion short reads are obtained in single run (Reis-filho, 2009)
  5. 5. Next generation sequencing platforms
  6. 6. Sanger sequencing • ddNTPs – Chain Termination • Sanger sequencing (1977) – Chain termination method – Sequencing by synthesis – Dideoxy sequencing – Most common approach used for DNA sequencing – Nobel prize – 1980 – Considered as the GOLD STANDARD
  7. 7. Maxam - Gilbert sequencing ((1977) Chemically cleavage Method  No need of DNA synthesis  Treatment of DNA with certain chemicals  DNA cuts into fragments  monitoring of sequences
  8. 8. NGS Workflow Sample • Source of sample (DNA, RNA) • Qualify and quantify samples Library • Prepare platform specific library • Qualify and quantify library Sequenc ing • Perform sequencing run reaction on NGS platform Analysis • Application specific data analysis pipeline Grada et al.;2013
  9. 9. Cluster Amplification: Bridge PCR Used by Illumina – DNA fragments are flanked with adaptors – Flow cell is coated with primers complementary to the two adaptor sequences – Isothermal amplification – Clusters of DNA molecules are generated on the chip – Each cluster is originated from a single DNA fragment, and is thus a clonal population
  10. 10. Cluster Amplification :Emulsion PCR Used by SOLiD, Ion torrent and pyrosequencig – Fragments attached with adaptors – One PCR primer is attached to the surface of a bead – DNA molecules are amplified on the beads within a water –oil emulsion – Each bead bears clonal DNA originated from a single DNA fragment – Beads placed into the wells of sequencing chips – One well, one bead
  11. 11. Roche (454) GS FLX sequencer Principle –Based on pyrosequencing principle –Sequencing by synthesis –Detection of released pyrophosphate Procedure 1. DNA fragmentations and adaptor ligation 2. Emulsion PCR 3. Beads are placed into wells of PTP 4. Slide is flooded with any one of the four nucleotide (Metzker et al., 2010) Light signal recorded by camera
  12. 12. Illumina Solexa Analyzer • Major NGS platforms • Reads of 100-150bp • Chemistry – Fragment of DNA – Sonication – Nebulization – RE digestion • Library preparation – Bridge amplification • Sequence by synthesis • Data analysis http://res.illumina.com/documents/products/techsp
  13. 13. Illumina Solexa Analyzer Determine first base • Through CRT strategy • Which sequences the template strand one nucleotide at a time through progressive rounds of base incorporation, washing, imaging • Fluorescently labeled 3’-o-azidomethyl-dNTPs are used to pause the reaction, enabling removal of unincorporated bases and fluorescent imaging to determine the added nucleotide • Scan the flow cell with a CCD camera, the fluorescent moiety and the 3’ block are removed, and the process is repeated http://res.illumina.com/documents/products/techsp (Guo et al., 2008)
  14. 14. Illumina Solexa Analyzer http://res.illumina.com/documents/products/techsp
  15. 15. Applied Biosystems SOLiD Sequencer • Sequencing by oligonucleotide ligation and detection • Ligase enzyme used instead of polymerase • Procedure 1. Sample preparation 2. Emulsion amplification 3. Ligation reaction and imaging • Sequencer adopts the technology of two-base sequencing based on ligation sequencing • Each sequencing involves 5 rounds of cyclic steps 4. Data analysis Mardis;2013
  16. 16. Ligation and Imaging…Ligation chemistry Mardis;2013
  17. 17. Ligation and Imaging…Ligation chemistry Mardis;2013
  18. 18. Data analysis Mardis;2013
  19. 19. Ion Torrent PGM sequencer • Sequencing platform does not uses optic signals • Clustal amplification- emulsion PCR • Concept – Addition of a dNTPs to a DNA, polymer releases an H+ – Change in pH is detected by the semiconductor installed in each well of the chip – Each time the chip was flooded with one nucleotide after another, if it is not the correct nucleotide, no voltage will be found – Voltage change corresponding to each type of nucleotide in different way • Run time: 3 h Mardis;2013
  20. 20. Helicos True Single molecule sequencing • Considered as TGS • First NGS platform which used the concept of single molecule fluorescent sequencing • No amplification needed • Process •DNA fragmentation •Tailed with poly A • Hybridized to a flow cell surface containing oligo-dT •Sequencing-by- synthesis of billions of molecules in parallel (Thompson et. al., 2010)
  21. 21. Pacific Biosciences(SMRT™) The SMRT - considered as TGS • Zero-mode waveguides (ZMW) – Heart of the technology – Detect world’s smallest light volume • Each SMRT contain tens of thousand ZMW • DNA template- polymerase complex are immobilized at bottom of ZMW • Four dNTPs labelled with different dyes are added • Incorporation of nucleotides produces signal Metzker,M.L.(2010) DNA polymerase ZMW
  22. 22. Nanopore DNA sequencing • 4th -generation • Smallest Sequencer • Base detection without labels • Ist hand held nanopore DNA sequencer • Principle – DNA strand is pulled through the nanopore by the enzyme – Passing of molecule through the pore causes a temporary change in the potential between the two compartments for identification of specific molecule • Types Biological nanopore- transmembrane protein channels Example: alpha-hemolysin, Solid-state nanopore Example MinION (Wang et al., 2015)
  23. 23. Comparisons sequencing platforms Platform Amplificati on method Sequencing chemistry Signal detection method Run time Advantage Disadvantage Accuracy Sanger sequencing Vector based Chain termination Long read Expensive Impractical for larger genome seq 99.9% 454gsflx (Roche) Emulsion PCR Pyrosequenc ing Light 23h Long read Expensive Homopolymer error 99.9% Illumina( hi seq, mi seq) Bridge PCR Sequencing by synthesis Light 2 days Hts Expensive Short read Long run time 98% AB solid Emulsion PCR Sequencing by ligation Light 8 days Low cost / base Short read Long run time 99.9% Ion torrent Emulsion PCR Sequencing by synthesis Ph 3h Short run time Less expensive Longer read lengths Homopolymer error 99.9% Helicos No PCR TSMS 1gb/h No PCR 100% Pacbio No PCR SMRT SMRT Light 20 min Longest read Fast No PCR Low yield at high accuracy Expensive 100% http://www.molgen.mpg.de/899148/ows2013
  24. 24. METAGENO MICS NEXT GENERATION SEQUENCING GENOMICS TRANSCRI PTOMICS EPIGENOM ICS Whole genome seq Exome seq De novo seq Targeted seq RNA- seq Targeted RNA seq Nc RNA seq ChIP-Seq Methyl-Seq Ribosome profiling Microbiome Seq Applications of NGS
  25. 25. Three NGS levels: Which sequencing level should we choose?
  27. 27. SAGE (Serial Analysis of Gene Expression) Velculescu V.E.(1995),Science 270:484-487
  28. 28. Noncoding RNA Profiling and Discovery • ncRNA – RNA that are not translated into protein product. • Includes tRNA, rRNA, snRNA, snoRNA, siRNA, miRNA • siRNA & miRNA- Posttranscriptional regulation of gene expression • MPSS highly efficacious for discovery of novel miRNA • Sequence based approaches helps in detection of variants of known miRNA
  29. 29. Epigenetic Modification Analysis • Epigenetics – study of heritable gene expression that does not involve DNA sequence. • Two major type of modifications – DNA methylation – Histone tail modification • Power of NGS gave a boost to the study of (genome wide) DNA methylation • NGS -analysis clearly reveals all sequences , besides enriching methylated DNA sequences • Chemical tricks can be used to identify methylated nucleotides. • Sequencing both untreated and bisulphate-treated DNA will highlight the c- nucleotides that are methylated and not chemically converted resulting in a t when sequenced
  30. 30. Ancient DNA • Usually highly fragmented with average fragment lengths 51.3 bp • Isolated from fossils • Contains very different levels of contamination • Difficult to sequence  NGS technologies an ideal tool for ancient DNA research  Large data generated by NGS  Short read length (Knapp and Hofreiter, 2010)
  31. 31. • Uncovering the potential cause of the disappearance of the honeybee • To detect the cause of an infectious disease Cox-Foster,2007
  32. 32. Whole Genome Sequencing • Whole genome de novo sequencing – Process sequencing fragments of DNA and assembling them to make a full-length genome without referring to any previous information of available sequences for the same species • Comparison between cynomolgus(CE) and Chinese rhesus(CR) macaques • Abundant genetic heterogeneity • Whole genome sequencing helps to quantify introgression influence • High degree of sequence similarity with human disease gene orthologs Guangmei Yan et. al (2011), Nature Biotechnology; p:1019-1023
  33. 33. Genomic prediction in livestock using NGS • NGS- explore relationship between genetic and phenotypic diversity with high resolution https://doi.org/10.12972/jabng.2017
  34. 34. challenged control Selection of Disease Resistant Animals For Breeding • Disease resistance and immune response are quantitative traits • Direct selection based on recovery after infection is not a practical way of disease resistance breeding • Focusing on one bacteria or virus cannot improve the disease resistance to multiple pathogens • Genetic basis of general resistance to multiple infectious diseases, and identification of indicator traits that can be used in breeding programme • Two groups : 1. Not infected(control) 2.Infected or challenged/immunised/cell culture Extract RNA Separation of m-RNA RNA – sequencing and analysis Detection of DE genes
  35. 35. • Genome sequencing facilitates the identification of genomic variation within different individuals by sequencing and comparing the data of individual genomes with reference genomes. • After successful alignment to a reference genome, SNPs are identified(heritable variations in animal genome) • Recent advances in NGS of DNA have enabled the systematic identification of CNVs at a higher resolution and sensitivity • SNP’s can be used to  Analyze genetic diversity  Characterization of genetic population structure  Genetic mapping, QTL mapping  Marker Assisted Selection and Breeding  Detection of genetic diversity among populations of a species has important effect on adaptive potential of a species Variant (SNP) Detection (Moore et al., 2011)
  36. 36. Assembled domestic animal genomes Bovine Womack, 2006 Water buffalo Michelizzi et al., 2010 Porcine Mote and Rothschild, 2006 Sheep Cockett, 2006 Horse Chowdhary and Raudsepp, 2006 Chicken Burt, 2006 Canine Galibert and Andre, 2006 Feline Murphy, 2006Pareek et al., 2011 Royal Bengal tiger genome sequenced The high coverage genome sequencing and identification of genome variants in Bengal tiger were carried out by scientists from the Centre for Cellular and Molecular Biology (CSIR-CCMB) and a Hyderabad-based private company.
  37. 37. Future challenges  Defining variability in many human and animal genomes  Analysis of vast production of sequencing database through advanced bioinformatics tools
  38. 38. Conclusions • Recently, HT-NGS technologies emerged as a potential research tool for the development of animal genomes research • Major role in genetic improvement of animal health and productivity in near future • NGS can be used to study functional as well as comparative genomics • Provided insight into epigenomic study • Helpful in providing information about evolution

Notas do Editor

  • Emulsion PCR
    Fragment DNA and attache adapters.
    PCR is carried out in a oil mixture containing beads and reagents.
    Each of the beads has a small adaptor that is complementary to the adaptor attached to the DNA fragment.
    Once the DNA is added to the emulsion ideally you get one fragment attached to each bead.
    You then carry out the PCR reaction which results in multiple clonal DNA fragments on each bead.
    The beads are then lowered on plates containing millions of wells roughly the size of each bead.