bioinformatics

1. Introduction, Medical Application and Recent Advances in Molecular Biology and Bioinformatics Refresher Course in Molecular Biology and Bioinformatics, 2018 Padmini Narayanan, Ph.D. pnarayan@bcm.edu

2. Learning Objectives Why do you need to attend this series? What can you expect from this course? How can you translate this knowledge in to better patient care? To provide a solid introduction to molecular biology with an emphasis on techniques and bioinformatic tools that are currently used to investigate the molecular mechanisms and its application to diagnose and treat diseases. To introduce and give context to all the following lectures of this course

3. From: Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical sciences Brief Bioinform. 2016;19(2):286-302. doi:10.1093/bib/bbw114

4. Parasitology Physiology Biophysics Developmental Biology Bio-Informatics Molecular Diagnostics Structural Biology Biochemistry Gene Therapy Molecular Epidemiology Pharmacology Virology Genetics Molecular Biology Enzymology

5. lindsaydigital.com • Molecular Medicine Molecular Insights • Identify pathology • Find inheritance of disease • Find ‘candidate gene’ • Screen for mutations Applications • Improved diagnosis of disease • Earlier detection of genetic predisposition to disease (BRCA1) • Rational drug design • Gene therapy • “targeted/custom drugs"

6. Parasitology Physiology Biophysics Developmental Biology Bio-Informatics Molecular Diagnostics Structural Biology Biochemistry Gene Therapy Molecular Epidemiology Pharmacology Virology Genetics Molecular Biology Enzymology Translational Bioinformatics

7. Diseases are inherently complex with multitude of interactions on different levels – genomic-environmental-transcriptome-etc. Network analysis approach offers sophisticated means for multidimensional omnics data integration. Its integrative informatics – pulling and curating information from various “-omnics” – identifying functional subnetworks in complex disease phenotypes – prioritizing candidate genes (GWAS) Exploring biochemical networks for identifying drug combinations and off target effects PPI networks – revel novel pathways – molecular underpinning of diseases Translational Bioinformatics – Clinical Significance

8. Translational Bioinformatics – Clinical Significance Using this approach – new methods/ models can be developed Better understanding of different aspects of the disease progression Generate new diagnostic tools – biomarkers – prognostic and drug targets Valid hypothesis generation , targeted therapy – personalized medicine

9. RESOURCES The Cancer Genomic Atlas (TCGA) Therapeutically Applkicable Research to generate Effective Treatments (TARGET) Cancer Target and Driver Discovery Network (CTD2) NCI Genomic Data commons (GDC)

10. PROCESSES, TOOLS AND APPLICATION

11. Replication Gene and Gene regulatory units Transcription Translation Posttranslational modifications Splicing Transport, Stability Structure, Transport, Stability Interactions Activity Downstream signaling Structural support https://www.dnalc.org/resources/animations/

12. Exceptions to Central Dogma of Molecular Biology RNA dependent RNA synthesis DNA RNA Protein Reverse transcription GENOTYPE PHENOTYPE

13. Increasing genome repertoire and functional diversity

14. A genome is an organism’s complete set of • DNA, including all of its genes • Each genome contains all of the information • needed to build and maintain that organism • In humans, a copy of the entire genome has • more than 3 billion DNA base pairs and is • contained in all cells that have a nucleus DNA- Structure and Genomes DNA structure and Genomes Aug 10th 2018 Dr. YasminkaJakubek

15. Human Genome, 3x109bp packaged into chromosomes • <3 % is expressed as proteins!! • ~80% is transcribed • What does the transcripts do?? • remaining 98–99% (non-coding regions) holds structural and functional relevance  Regulatory function http://www.sciencemag.org/news/2012/09/human-genome-much-more-just-genes

16. DNA Replication • ~160 proteins are involved in replication • Numerous genetic diseases result from mutations in these proteins or from errors in DNA replication or repair • e.g. recQ Helicases: Premature Aging, cancer predisposition (Werner’s syndrome) DNA replication and PCR methods Aug 17th 2018 - Dr. Aparna Krishnavajhala

17. PCR: in vitro DNA Replication PCR Applications • Molecular Biology and Genetics Research • Clinical Applications • Forensic Sciences • RT-PCR: Reverse Transcription PCR • Real time quantitative-PCR (qPCR) • Nested PCR • PCR Arrays • Multiplex PCR 1st cycle 2nd cycle 30th cycle 22=4 copies 23=8 copies 231=2 Billion copies

18. DNA Sequencing • Very important tool for biologists • Sequence of genes • Positioning of genes • Sequences of regulatory regions Applications: • Health Care • Diagnostics • Forensics (DNA fingerprinting) • Agriculture • Research Next generation DNA sequencing methods Aug 24th 2018 Dr. Devon Marie Fitzgerald

19. Sanger Sequencing – base by base sequencing of a locus – 1kb per run • DNA-microarrays – hybridization of the DNA sample with a set of pre-defined oligonucleotide probes distributed across the entire genome or enriched around regions of interest • Next-generation sequencing (NGS) - fragmentation of the genomic DNA into pieces that are subsequently sequenced and aligned to a reference sequence. Allows detection of novel changes • Whole Exome Sequencing (WES)– specific region – sequencing of all variants in the coding regions – reveals protein affecting modifications • Whole Genome Sequencing (WGS) of an individual – identify all rare coding and non-coding variations. Technologies and methods to identify genetic variants - Simple Nucleotide Variants (SNVs) & Structural variants (SVs)

20. From: Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical sciences Brief Bioinform. 2016;19(2):286-302. doi:10.1093/bib/bbw114

21. Questions • Is DNA the only genetic material? • Is DNA sequence/gene alone responsible for heritable traits? • Can heritable changes take place without changes in DNA Sequence?

22. What is epigenetics? • DNA methylation: • CpG islands: • transcription starting sites (UTR-areas): hypo-/hypermethylation • imprinting (X-inactivation): hypermethylation • Repetitive sequences: • Transposable elements: hypermethylation • Histone modifications: • Acetylation: HAT/HDAC- enzymes • Methylation: H3K4me3: near the transcription start sites • Phosphorylation, Ubiquitylation, Sumoylation • Mitotically or meiotically heritable changes in gene expression that don’t involve a change in DNA sequence. • Macromolecules that bind and functionally affect the metabolism of the DNA • Dependent on cell/tissue type!

23. Epigenomics Epigenomics – methylation and histone modification Aug 31st 2018 Dr. Jacob Junco

24. Epigenomics • The Encyclopedia of DNA Elements (ENCODE) Program that screens the entire genome and map every detectable functional unit in the database. •  identify signature patterns, such as DNA methylation, histone modification and transcription factors, suppressors that bind the specific region. •  88% of trait associated variants detected with GWAS fall in non-coding regions, ENCODE will tremendously impact their assessment and phenotype-related interpretation

25. DNA Repair, Genomic Instability • Mutation in a DNA repair gene can cripple the repair process (Werner, Rothmund Thomson) • Repair mechanisms restore DNA to its original state • Application: • Disease pathology (Cancer, accelerated aging etc) DNA repair, recombination and genomic instability Sept 7th 2018 Dr. Devon Marie Fitzgerald

26. Manipulating DNA molecule – study effect of genes – develop novel medicine Recent advances – rather than studying the DNA taken out of the genome –able to modify genes directly in their endogenous context – any organism Elucidate their effect in relevant environment – functional organization of the genome at the system levels – identify causal genetic variations. Genome editing A new era in molecular biology Just CRISPR it!!!

27. Targeted Genome editing and CRISPR/Cas9 A new era in molecular biology Genome engineering with Cas9 nuclease Origin: Bacterial immune system • Guide RNA • Cas9 (CRISPR associated endonuclease) • Make precise modifications in target DNA (HDR) • Generate knock-outs (NHEJ) • Activate or repress target genes (promoter binding) • Genome wide screens (disease/pathway associated novel genes) Targeted genomic editing CRISPR Sep 14th 2018Dr. Hyun Hwan

28. CRISPR applications and Animal Models Application of genomic eng - causal genetic mutation – Rapid identification of epigenetic variants associated with altered biological functions or disease phenotype – effectively recapitulated in animal and CRISPR application and Animal Models Sep 21nd 2018 Dr. Sirena Soriano Genetic and epigenetic control of cells with genomic engineering – Broad rage of application. Hsu. P, et al. Cell 2014 June5, 157(6).

29. Gene Expression: Transcription, Alternative splicing, RNA Seq Regulation of gene expression is the critical link between the genome and cellular morphology/physiology • Transcription • Transcriptional Factors • Transcriptional Regulation Sep 28th 2018 Dr. Tao Ling • 90% of human genes are alternatively spliced!

30. Gene Expression: Non-Coding RNA and Personalized Medicine • A non-coding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein. • Non-coding RNAs: Junk or Critical Regulators in Health and Disease? • Prader–Willi syndrome (snoRNA-HBII52) • Alzheimer's disease (lncRNA-BACE-AS) • Cancer (miR200) RNAi therapeutics – potent –specific mechanism –targeting gene expression – siRNA bound o RNA induced silencing complex(RISC) mediated target mRNA degradation using endonucleases (Arg). Pathway present in all mammalian cells. First-in-Humans Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement Cancer Discovery April 2013 3; 406

31. Gene expression – Non-coding RNA and personalized medicine. Breaking the genetic enigma Oct – 5th 2018 – Dr.NarayanSastriPalla

32. Translation and Protein Function Prediction and detection • mRNA to Protein • Single nucleotide change can alter codon and possibly aminoacid • Change in amino acid sequence causes changes in – 3-D structure of protein – Defective protein folding – Protein function • Cystic fibrosis – Misfolded CFTR protein – Protein retained in ER • Huntington disease – Trinucleotide repeats – Multiple CAG repeats • Antibiotics often target bacterial • Translation

33. From: Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical sciences Brief Bioinform. 2016;19(2):286-302. doi:10.1093/bib/bbw114

34. Protein Detection Extraction of proteins Separation of proteins Identification of proteins Quantification of Proteins Western Blot Immunohistochemistry/Immunofluorescence ELISA/EIA Immunoprecipitation Mass-Spectrometry X-Ray Chrystallography

35. Tissue Preparation for IHC

36. Immunohistochemistry Signal amplification is not possible Suitable for high abundant ptn. Moderate signal amplification – commonly used. Avidin-biotin complex detection Substrate usually Horse Radish peroxidase (HRP) or alkaline phosphatase (AP) or the primary or secondary ab will be conjugated with fluorophore.

37. Immuno-colocalization of GP130 surface receptor with Lipid rafts in Pediatric AML blasts MLLM-13 Kasumi THP-1 K562 K61 NB4 HS5 260 125 70 38 Caveolin-1 15 Translation, function prediction and detection of proteins Oct 12th 2018 Dr. Nikhil Jain GP130 Surface staining observed using flowcytometry

38. Protein Modification: Localization and activity • Enzymes, Receptors, Cytokines, Hormones etc. • Translation, Transport and activity • Metabolic disorders • Types of protein modification Molecular Biology Techniques • Activity Assay • Identification of Post-translational modification Protein Modification – Localization and activity Oct 19th 2018 Dr. Poonam Sarkar

39. Cell cycle and chromosomal Instability • Historical context • Preservation and propagation of gene associated diseases • Molecular biology techniques used to these disorders • Molecular biology application in treatment • Introduction to relevant Bio-informatic tools Oct 26th 2018 Dr. RenukaRamankutty Menon

40. Based on isolation of DNA sequence of interest to obtain multiple copies invitro • Insertion of genetic information in to replicating vehicles • Plasmids, viral vectors, BAC etc. • Different techniques of cloning and vectors tailored to the final application. • Lecture – Structure of plasmids, cloning sites, restriction enzymes- cloning softwares and clone a gene in a vector. Molecular Cloning and its application Nov 2nd 2018 Dr. ParamahamsaMaturu

41. Genome-wide association study • A genome-wide association study is an approach that involves rapidly scanning markers across the complete sets of DNA, or genomes, of many people to find genetic variations associated with a particular disease. • Once new genetic associations are identified, researchers can use the information to develop better strategies to detect, treat and prevent the disease. Such studies are particularly useful in finding genetic variations that contribute to common, complex diseases, such as asthma, cancer, diabetes, heart disease and mental illnesses. http://www.genome.gov/20019523

42. GWAS- Population Genetics • Introduction to genome-wide association studies (GWAS), clinical relevance of GWAS of complex disease • Introduction to relevant databases to identify haplotype blocks and performing genetic and epigenetic profiling or susceptibility loci • Experimental techniques to determine underlying mechanisms of susceptibility loci • Walkthrough of a leukemia-specific post-GWAS analysis using HaploReg and RegulomeDB

43. Potentials of GWAS GWAS population Genetics Nov 9th 2018 Dr. Vince Gant

44. So by the end of this series ….. “Must be a clinical fellow." Well! this meme will be obsolete…..

45. Bioinformatics • OMIM • Gene Card • Gene • NCBI Primer Design • UCSC Genome Browser • HPRD • ExPASy

46. OMIM • Online Mendelian Inheritance in Man® • Compendium of genes & genetic phenotypes • Contain information on all known Mendelian • disorders (~15,000 genes) • Relationship between phenotype & genotype • Initiated 1960s by Dr. Victor A. McKusick • McKusick-Nathans Institute of Genetic • Medicine, JHU • www.omim.org

47. Type gene or disease name to get further details

48. *176730 INSULIN; INS Alternative titles; symbols PROINSULIN Other entities represented in this entry: INS-IGF2 SPLICED READ-THROUGH TRANSCRIPTS, INCLUDED; INSIGF, INCLUDED HGNC Approved Gene Symbol: INS Cytogenetic location: 11p15.5Genomic coordinates (GRCh37): 11:2,181,008-2,182,438 or.. New Gene-Phenotype Relationships -:3+• Table of Contents for *176730 Title Gene-Phenotype Relationships Text Description Gene Structure Mapping Gene Function Biochemical Features Molecular Genetics Animal Model History Allelic Variants Table View See Also References Contributors Creation Date Edit History External Links for Entry: Genome • DNA • Protein • Gene Info ...Clinical Resources • Variation Animal Models Location Phenotype Phenotype MINI number 125S52 613370 3.10.5.5 Hypexproinsulinenta Maturity-onset diabetes of the voting, type 10 Phenotype mapping key 606176 616214 Diabetes mellitus, insulin-dependent, 2 Diabetes mellitus, permanent neonatal 3 3 3 3 TWYT

49. Gene • http://www.ncbi.nlm.nih.gov/gene • F;72: NCBI Resources 1:vi How To NI Gene Gene No. Advanced Help Gene Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes, and links to genome-, phenotype-, and locus-specific resources worldwide. Using Gene Gene Tools Other Resources Gene Quick Start Submit GeneRIFs 1-10moloGene FAQ Submit Correction OMIM DowniaadIFTP StatisticsRefSeo RefSeq Mailing List BLAST RefSeqGene Gene News 13 Genome Workbench UniGene FactsheetSplignProtein Clusters

50. 1 of 64 Next > Last » Page Aliases Location Chromosome 1, ER-alpha, Esr • Esrl estrogen receptor 1 [Rattus NCBI Resources 17.1 How To 1,71 Sign in to NCBI Gene V Help Hide sidebar » Send to: 2 Advanced Display Settings: l,71 Full Report re, NCBI Resources El How To 17 Sign in to NCBI estrogen receptor rat X Gene Gene Create alert Advanced Help Send to: (=I Gene sources Genomic Hide sidebar » Filters: Manage Filters Display Settings: 0 Tabular, 20 per page, Sort by Relevance Categories Alternatively spliced Annotated genes Non-coding Protein-coding Pseudogene Results: 1 to 20 of 1271 0 See also 13 discontinued or replaced items. Name/Gene ID Description Results by taxon Top Organisms [Tree]Homo sapiens (644) Mus musculus (300) Rattus norvegicus (294) Gene A Table of contents Summary Genomic context Genomic regions, transcripts, and products Bibliography Variation Pathways from BioSystems Interactions General gene information Markers, Homology, Gene Ontology General protein information NCB! Reference Sequences (RefSeq) Related sequences Additional links Related information Unactive previous page of results Esrl estrogen receptor 1 [ Rattus norvegicus (Norway rat) ] Gene ID 24890, updated on 25-Jul-2015 Esr1 provided by RGD estrogen receptor 1 provided by RGD RGD:2581 Ensembl:ENSRNOG00000019358; Vega: OTTRNOG00000001366 protein coding PROVISIONAL Rattus norvegicus Eukaryota: Metazoa; Chordata; Craniata; Vertebrata: Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae, Murinae; Rattus Esr; ER-alpha: RNESTROR acts as a transcriptional activator when bound to estrogen; may play a role in myocardial regulation IRGD, Feb 2006] Annotation category suggests misassembly Summary Official Symbol Official Full Name Primary source See related Gene type Ref Seq status Organism Lineage Also known as Summary Annotation