BIOLOGY 305 1st Edition Lecture 27 Outline of Last Lecture I. Transposable ElementsII. McClintock Tranposon DiscoveryA. The Ac-Ds SystemB. Examples of transposable elementsIII. Genetics of DevelopmentA. 5 classes of Mutations affecting Drosophila Embryonic DevelopmentB. Maternal Effect and Hox GenesIV. Genetic Model Organisms for DevelopmentV. Genetic ScreensOutline of Current Lecture I. Sequencing Large GenomesII. Genome AnnotationIII. TranscriptomesIV. Chromatin Immunoprecipitation (ChIP)V. Key Vocabulary and Sample QuestionsCurrent LectureI. Sequencing Large GenomesWhole Shotgun Sequencing (WGS) – “sequence first, map later”A method used for sequencing long DNA strands. It is a rapidly expanding process that is semi-random in terms of firing patterns. The long sequences have to be divided into smaller gradments and re-assembled to give overall sequenceRecall: Sanger Sequencing was only good for relatively short strandsIn WGS, every sequence has to be unique: sequenced both ends of a fragment of DNAsequencing both ends of the same fragment is more difficult for larger genomestandem repeats on many chromosomes = we wouldn’t know which should be assignedKnowing the anti-parallel property and length of fragment helps reconstructionOrdered Clone Shotgun Sequencing (Hierarchical Shotgun) – “map first, sequence later”1) Physical map consisting of a collection of genomic library clones ordered along genome map2) Each clone is sequenced after map is assembledSequencing Large Genomes:1) Broken up randomly into randome clones of Genomic DNA2) Randomly sequenced using chain termination to obtain short-reads (recall genome libraries and Sanger)3) Obtain multiple overlapping reads for target DNA by many rounds of fragmentation and sequencing4) Computer assembles the overlapping ends ina continuous sequenceGenerating a physical map requires a large amount of workGenomic location is known immediately, thus sequence assembly is easierII. Genome Annotation - How do we make sense of it?The first thing that always has to be done is: Annotation – to identify genes in DNA sequencesIdentifying protein coding sequences:1) Find Open Reading Frames (ORFs)Contains a Start and stop codon – candidates are protein coding genes but not all of them encode anything at all2) Find cDNA/EST (DNA obtained from an RNA template) sequences3) Confer sequence and compare to known genes in other organismsNote: This does not prove similar functions, only suggestsAnnotations are only based on data-source and not experimentation and are subject to being erroneous. ~25,000 genes estimated for humans, 3% are coding, 45% are repetitive#1: How to find an ORF?1) Find all ATGs on DNA2) Make sure they are in-frame (multiples of three)3) An open-reading frame can be as short as one amino acid#2: How to find cDNA/EST Sequences?cDNA only have exons while genomic DNA shows where transcription startsexons and introns are created, and where transcription is terminatedEST is expressed sequence tags, easy to sequence these at the ends of RNA#3: Confer sequence and compare to known genes in other organismsMake sure you get practice in how to analysis these kinds of problems (see lecture)III. TranscriptomesSome terminology:Genome – all DNA of genesProteome – all proteinMetabolome – all metabolitesInteractome – all interactions of proteinEpigenome – all chromatin and histone modifications of a genomeTranscriptomes are all the RNAs of an organismWant to find the function of a gene? The transcriptomes helps you uncover thisDNA Microarray Techniques:Basic:- Spot DNA in all the arrays on the chip, each drop has various DNA sequences in them- The samples are incubated- Samples are indicated by color, when RNA sample hybridizes with the DNA on array:Green – lots of RNA sample 2Red – lots of other RNA sample 1RNA sequencing- Total RNA (transcriptome) extraction- RNA is sheared into short fragments- Sequenced using Next Gen sequencing- Count sequencing reads mapping to a gene:The resulting spikes correspond to exons IV. Chromatin Immunoprecipitation (ChIP)1) Lyse cell with sonification or enzyme digestion2) Isolate and purify this locus bound by the protein3) Use antibodies that are able to recognize the target protein in the chromatin, pull this all out4) Use PCR to analyze the sequence boundedCan analyze the purified DNA with PCR, MicroArrays, or Sequencing techniquesChIP-sequencing: Genome WideChIP can also be applied for genome wide analysis by combining with Next Gen sequencing (seq)Transcription and histone modifications can be shown by ChIP-seq Evidence of protein binding to the locus is shown by spikesV. Key Vocabulary and Sample QuestionsGenome – the entire complement of genetic material in a (haploid) chromosome setGenomics – the study of genomes in their entiretyGenome project – the sequencing of an entire genome for a given model organismBioinformatics – the analysis and annotation of the information content, such as genes, binding sites for proteins, and RNAsComparative genomics – the comparison of genomes of different organismsFunctional genomics – the use of high-throughput methods to study gene action and gene networksSequence reads – a read of about 600 bp of a single sequencing reaction10-fold coverage – a convention where all the fragments must be sequenced 10 times to get a reliable sequencegenomic library – generated by fragmenting DNA and ligating into cloning vectors, which must be done before sequencingDNA polymorphisms – sequence variations between individuals within the same speciesDNA markers – a DNA fragment of known size and location with identifiable variantsPhenotype = presence of band on SB or PCR, etc…SAMPLE QUESTIONS:1) What is the biggest challenge for the Human Genome Project?A. Obtaining DNA samplesB. Sequencing DNAC. Understand the information encoded by the genomeFind all open reading frames in the sequence below.2) You performed RNA-seq experiment using rice total RNA. The picture below shows existing gene annotation and result of your RNA-seq. What is your conclusion?A. Region 1 may contain a previously unidentified intronB. Region 2 may contain a previously unidentified intronC. Region 3 may contain a previously unidentified intronD. Region 4 may contain a previously unidentified intronE. All of the aboveANSWERS:1) CObtaining a DNA sample is relatively easy. As we have seen with WGS and HS, sequencing a genome is