Genome Sequencing: Harmonia axyridis

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Genome Sequencing: Harmonia axyridis. Isabel Risch University of Memphis W. Harry Feinstone Center for Genomic Research May 28, 2013-June 14, 2013. Ladybugs (Ladybirds). Tennessee state insect Coccinella septempunctata Seven-Spotted Lady Beetle - PowerPoint PPT Presentation

Text of Genome Sequencing: Harmonia axyridis

Gene Sequencing: H. axyridis

Genome Sequencing: Harmonia axyridisIsabel RischUniversity of MemphisW. Harry Feinstone Center for Genomic ResearchMay 28, 2013-June 14, 2013

Ladybugs (Ladybirds)Tennessee state insectCoccinella septempunctataSeven-Spotted Lady BeetleNative to North America; being outcompeted by HarmoniaHarmonia axyridisAsian/harlequin lady beetleLarge coccinellid beetleDome-shaped; smooth transition between head and thorax/abdomenAdults colored anywhere from yellow to bright redSpots on back can be anywhere from zero to twentyNative to Asia; introduced to North America and Europe in order to control aphid populations; now crowding out other species (invasive)Carries a fungus that kills other species of ladybugsHarmonia creates the chemical harmonine which prevents the fungus from infecting it

2Genome: What Is It?An organisms hereditary information, coded in DNA/chromosomes; in eukaryotes, includes introns and exonsChromosomes: DNA wrapped around histones

Human Chromosome PaintingGenome: What Is It Made Of?DNA (deoxyribonucleic acid) Called the molecule of life Codes for all proteins that make cells (life) possibleMade of deoxyribose, three phosphate groups, and a nitrogen baseDouble-stranded molecule; covalent bonds between ribose/phosphate backbone on outside; hydrogen bonds between nitrogen bases on insideAllows for the breaking of hydrogen bondsreplication and expression through RNABases: Adenine, Thymine, Guanine, Cytosine; A-T, G-COrder of nitrogen bases codes for specific amino acids polypeptide chains proteinIn eukaryotes, contains both introns (non-coding sections) and exons (coding sections)

Genome: What Is It Made Of?DNA and HeredityHeredity: the passing of traits from one generation to the next basis of genetics and evolutionDetermined by genes on chromosomes; variations of a gene are allelesSexually-reproducing animals get two alleles (one from each parent)Mendels Law of SegregationAlleles express themselves as phenotypic traits; thus, DNA determines heredity

Genome: What Can We Do With This Information?By determining the sequence of genomes, we canCompare them to other genomesStudy phylogeny and evolutionUse them to understand diseases and better create potential treatments; also better predict the bodys response to certain treatmentsGenetic diseasesSomatic diseasesUse them for forensic scienceResearch deeper into genetic engineering of plants and animals (biotechnology)Genome MappingCan be done once a genome is sequencedDetermines the physical order of the sequence features of the entire DNA of an individualPlaces certain DNA fragments onto chromosomes by identifying the fragments Identify by certain markers or by the exact base pair sequence of DNATraditional maps mapped millions of base pairs at once (low resolution), but modern ones can map in SNPs (one or two base pairs at a time) for higher resolutionsCan be used to identify a certain genetic marker with a certain diseaseSomatic diseasesEx: cancer can occur when a tumor-suppressing gene is inactivated or blocked; genome mapping can be used to identify the genes and research ways to reactivate themGenetic diseasesEx: sickle cell anemia is related to a mutation in the beta hemoglobin gene

DNA Sequencing: BackgroundSanger MethodUsed to determine nucleotide order in DNARapid DNA sequencingUses modified, labeled nucleotides to stop DNA strand elongation at specific basesScientists treat each DNA sample with one labeled baseDNA can then be run on a gel and tracked to where it was terminated; nucleotides separated by size and nucleotide typeResults photographed on an X-ray or gel imageDye-terminator sequencing: revised methodUses fluorescent dyes to visualize all bases on one lane

DNA Sequencing: BackgroundIllumina TechnologiesNext-generation sequencingA single strand of DNA fragment provides a template for the DNA to be re-synthesizedSignals are emitted and interpreted by the sequencing machineUnlike Sanger, next-gen can be applied to millions of base pairs at once via a flow cellFragmented reads are then re-assembled by alignment whole genomeMiSeqPersonal tabletop sequencerCapable of many of the functions of a large sequencerUses fluorescence and LED light while previous machines used lasersCheaper now many universities can afford sequencers

DNA ExtractionThe process of separating pure genomic DNA from the rest of the contents of cells and tissuesSteps:Lysing cells (breaking them to get to DNA)Removing contaminants from DNA (proteins, RNA, lipids, etc.)Pelleting DNA (precipitating and compacting it to separate it from everything else)Washing away solutions used to purify DNAGenome SequencingThe process of determining the nucleotide order of a specific genomeDNA extractionDNA prepTagmentation, amplification, etc.Run on a sequencerAlignment and re-assemblyGenome Sequencing HarmoniaWhy we sequenced it:To better understand the insect and other beetles close to itWhat we used to sequence it:G Biosciences DNA extraction/prep kits Illumina sequencer (MiSeq)Blue Pippin to run gels and size selections QuBit to measure DNA concentrations in samplesGenome had very low diversity; difficult to sequenceMay be due to transposon activity/repetitive elements in the genome

Steps of SequencingDNA ExtractionHarmonia pupa homogenizedLyse cells reach DNA insideProteinase K addedBreaks down proteins surrounding the DNA (purifies)Chloroform addedPrecipitates waste from DNADNA Stripping Solution addedStrips DNA of any more wastePrecipitation Solution addedPrecipitates wasteIsopropanol addedPrecipitates DNA so it can be separated from other parts of mixtureEthanol washWashes DNA to further purify (remove excess salt)

Steps of SequencingPaired End PrepFollowed Nextera XT DNA Prep Kit (Illumina, San Diego, CA)TagmentationDNA is fragmented and tagged (adapters added to DNA ends) allows DNA to be PCR amplifiedPCR AmplificationDNA is amplified in a polymerase chain reactionAmplification: DNA is replicated many times over so the sequencer can read itPCR Clean-upDNA is purified using AMPure Beads (unusable bits of DNA are washed out)Library NormalizationMakes sure that the DNA quantities from each sample are equal in the final pooled library

Steps of SequencingMate Pair PrepFollowed the Nextera Mate Pair DNA Prep Kit (Illumina, San Diego, CA). Two versions of the mate pair were runGel-plus/size selectionUsed a Blue Pippin Prep machine (Sage Sciences, Beverly, MA)Yielded fragments 10kb-17kbGel-freeYielded 3kb-15kb fragments

Steps of SequencingMate Pair Prep: Gel-FreeTagmentationStrand Displacement ReactionPolymerase is used to fill gaps in DNA caused by tagmentationAMPure PurificationUsable DNA binds to AMPure Beads; anything unwanted in the solution, including small DNA fragments, is washed away

Steps of SequencingMate Pair Prep: Gel-FreeCircularizationFragments are circularized with blunt-ended ligationExonuclease DigestionAny remaining linear DNA is broken down, removed from the circularized fragmentsFragmentation of Circularized FragmentsCircularized DNA is sheared to smaller fragments by sonication

Steps of SequencingMate Pair Prep: Gel-FreePurification of Mate Pair FragmentsUsable DNA fragments bind to streptavidin beads; everything else is washed awayUsable DNA= fragments containing biotinylated adaptersEnd Repair/A-TailingOverhangs from DNA shearing are blunted3 overhangs are removed; 5 are filled in with polymeraseAn A nucleotide is added to the 3 endsSteps of SequencingMate Pair Prep: Gel-FreeAdapter LigationIndexing adapters are added to the ends of the fragmentsContain a T nucleotide that ligates to the A tailPrepares the fragments for amplification and flow cell hybridizationPCR AmplificationPCR Clean-up

Steps of SequencingMate Pair Prep: Gel-PlusTagmentationStrand Displacement ReactionAMPure PurificationSize SelectionUsed a Blue Pippin Prep machine (Sage Sciences, Beverly, MA)Specific range of DNA fragment sizes are chosen and separated from rest of DNA10-17kbCircularizationExonuclease DigestionFragmentation of Circularized FragmentsPurification of Mate Pair FragmentsEnd Repair/A-TailingAdapter LigationPCR AmplificationPCR Clean-up

Steps of SequencingSequencing Paired EndsSample was diluted with hybridization buffer and paired-end sequenced in the MiSeq2x250 runSequencer reads 250bp at a timeRun yielded poor-quality data (low diversity)Spiked with PhiX, re-run

Steps of SequencingSequencing Mate PairsGel and non-gel libraries diluted to 2 nM with Tris-Cl 10 mM, pH 8.5 with 0.1 Tween 20 2nM of DNA from each library was pooledPooled library was diluted with 0.2N NaOH and hybridization bufferMixture was diluted again with hybridization buffer Placed on the MiSeq for mate pair sequencingRun yielded poor-quality data (low diversity)Sample was spiked with PhiX, re-runAssemblyFirst, DNA quality is charted and basic stats are reviewed (FastQC)Use charts to find which bases to trimTrim first and last bases (bad quality unusable)Aligned reads to reference genome (or similar genome in de novo assembly) in BWA (Burrows-Wheeler Aligner)BWA output files are imported into Integrative Genome Viewer (IGV)Overlaps in read sequences allow whole genome to be re-assembled

IGV: viewing depth of coverage and fragment lengths

Paired ends give 100x coverageMate pairs provide scaffoldResultsH. axyridis genome is about 300 million bp longAfter trimming, we ended with628,908 paired end reads4,038,064 singletons1,454,689 mate pair reads (non-gel)199,700 mate pair reads (gel)Low diversity suggests transposon activity in genomeGenome full of long A sequencesAcknowledgementsThanks to the W. Harry Feinstone Center for Genomic Research and especially the Sutter Lab for allowing me to intern with them

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