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 PresentationTRANSCRIPT
Genome Sequencing: Harmonia axyridisIsabel RischUniversity of MemphisW. Harry Feinstone Center for Genomic ResearchMay 28, 2013-June 14, 2013
Ladybugs (Ladybirds) Tennessee state insect Coccinella septempunctata
Seven-Spotted Lady Beetle Native to North America; being outcompeted by Harmonia
Harmonia axyridis Asian/harlequin lady beetle Large coccinellid beetle Dome-shaped; smooth transition between head and thorax/abdomen Adults colored anywhere from yellow to bright red Spots on back can be anywhere from zero to twenty Native 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 ladybugs
Harmonia creates the chemical ‘harmonine’ which prevents the fungus from infecting it
Genome: What Is It? An organism’s hereditary information,
coded in DNA/chromosomes; in eukaryotes, includes introns and exons Chromosomes: DNA wrapped around
histones
Human Chromosome Painting
Genome: What Is It Made Of? DNA (deoxyribonucleic acid)
Called the “molecule of life” Codes for all proteins that make cells (life) possible
Made of deoxyribose, three phosphate groups, and a nitrogen base
Double-stranded molecule; covalent bonds between ribose/phosphate backbone on outside; hydrogen bonds between nitrogen bases on inside Allows for the breaking of hydrogen bondsreplication and
expression through RNA Bases: Adenine, Thymine, Guanine, Cytosine; A-T, G-C Order of nitrogen bases codes for specific amino acids
polypeptide chains protein In eukaryotes, contains both introns (non-coding sections)
and exons (coding sections)
Genome: What Is It Made Of? DNA and Heredity
Heredity: the passing of traits from one generation to the next– basis of genetics and evolution
Determined by genes on chromosomes; variations of a gene are alleles
Sexually-reproducing animals get two alleles (one from each parent) Mendel’s Law of Segregation
Alleles express themselves as phenotypic traits; thus, DNA determines heredity
Genome: What Can We Do With This Information? By determining the sequence of genomes, we can…
Compare them to other genomes Study phylogeny and evolution
Use them to understand diseases and better create potential treatments; also better predict the body’s response to certain treatments Genetic diseases Somatic diseases
Use them for forensic science Research deeper into genetic engineering of plants
and animals (biotechnology)
Genome Mapping Can be done once a genome is sequenced Determines the physical order of the sequence features of the entire
DNA of an individual Places certain DNA fragments onto chromosomes by identifying the
fragments Identify by certain markers or by the exact base pair sequence of DNA Traditional 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 resolutions
Can be used to identify a certain genetic marker with a certain disease Somatic diseases
Ex: 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 them
Genetic diseases Ex: sickle cell anemia is related to a mutation in the beta hemoglobin gene
DNA Sequencing: Background Sanger Method
Used to determine nucleotide order in DNA Rapid DNA sequencing Uses modified, labeled nucleotides to stop DNA
strand elongation at specific bases Scientists treat each DNA sample with one labeled
base DNA can then be run on a gel and tracked to where
it was terminated; nucleotides separated by size and nucleotide type
Results photographed on an X-ray or gel image Dye-terminator sequencing: revised method
Uses fluorescent dyes to visualize all bases on one lane
DNA Sequencing: Background Illumina Technologies
Next-generation sequencing A single strand of DNA fragment provides a template for the DNA to be
re-synthesized Signals are emitted and interpreted by the sequencing machine Unlike Sanger, next-gen can be applied to millions of base pairs at once
via a flow cell Fragmented reads are then re-assembled by alignment whole
genome MiSeq
“Personal” tabletop sequencer Capable of many of the functions of a large
sequencer Uses fluorescence and LED light while previous
machines used lasers Cheaper– now many universities can afford
sequencers
DNA Extraction The process of separating pure genomic
DNA from the rest of the contents of cells and tissues
Steps: 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 DNA
Genome Sequencing The process of determining the
nucleotide order of a specific genome DNA extraction DNA prep
Tagmentation, amplification, etc. Run on a sequencer Alignment and re-assembly
Genome Sequencing Harmonia Why we sequenced it:
To better understand the insect and other beetles close to it
What 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 samples
Genome had very low diversity; difficult to sequence May be due to transposon activity/repetitive elements
in the genome
Steps of Sequencing DNA Extraction
Harmonia pupa homogenized Lyse cells reach DNA inside
Proteinase K added Breaks down proteins surrounding the DNA (purifies)
Chloroform added Precipitates waste from DNA
DNA Stripping Solution added Strips DNA of any more waste
Precipitation Solution added Precipitates waste
Isopropanol added Precipitates DNA so it can be separated from other parts of mixture
Ethanol wash Washes DNA to further purify (remove excess salt)
Steps of Sequencing Paired End Prep
Followed Nextera XT DNA Prep Kit (Illumina, San Diego, CA) Tagmentation
DNA is fragmented and “tagged” (adapters added to DNA ends) allows DNA to be PCR amplified
PCR Amplification DNA is “amplified” in a polymerase chain reaction Amplification: DNA is replicated many times over so the sequencer
can read it PCR Clean-up
DNA is purified using AMPure Beads (unusable bits of DNA are washed out)
Library Normalization Makes sure that the DNA quantities from each sample are equal in
the final pooled library
Steps of Sequencing Mate Pair Prep
Followed the Nextera Mate Pair DNA Prep Kit (Illumina, San Diego, CA).
Two versions of the mate pair were run Gel-plus/size selection
Used a Blue Pippin Prep machine (Sage Sciences, Beverly, MA)
Yielded fragments 10kb-17kb Gel-free
Yielded 3kb-15kb fragments
Steps of Sequencing Mate Pair Prep: Gel-Free
Tagmentation Strand Displacement Reaction
Polymerase is used to fill gaps in DNA caused by tagmentation
AMPure Purification Usable DNA binds to AMPure Beads;
anything unwanted in the solution, including small DNA fragments, is washed away
Steps of Sequencing Mate Pair Prep: Gel-Free
Circularization Fragments are circularized with blunt-ended
ligation Exonuclease Digestion
Any remaining linear DNA is broken down, removed from the circularized fragments
Fragmentation of Circularized Fragments Circularized DNA is sheared to smaller
fragments by sonication
Steps of Sequencing Mate Pair Prep: Gel-Free
Purification of Mate Pair Fragments Usable DNA fragments bind to streptavidin
beads; everything else is washed away Usable DNA= fragments containing biotinylated
adapters End Repair/A-Tailing
Overhangs from DNA shearing are blunted 3’ overhangs are removed; 5’ are filled in with
polymerase An ‘A’ nucleotide is added to the 3’ ends
Steps of Sequencing Mate Pair Prep: Gel-Free
Adapter Ligation Indexing adapters are added to the ends of
the fragments Contain a ‘T’ nucleotide that ligates to the ‘A’
tail Prepares the fragments for amplification and
flow cell hybridization PCR Amplification PCR Clean-up
Steps of Sequencing Mate Pair Prep: Gel-Plus
Tagmentation Strand Displacement Reaction AMPure Purification Size Selection
Used a Blue Pippin Prep machine (Sage Sciences, Beverly, MA) Specific range of DNA fragment sizes are chosen and separated from rest of DNA
10-17kb Circularization Exonuclease Digestion Fragmentation of Circularized Fragments Purification of Mate Pair Fragments End Repair/A-Tailing Adapter Ligation PCR Amplification PCR Clean-up
Steps of Sequencing Sequencing Paired Ends
Sample was diluted with hybridization buffer and paired-end sequenced in the MiSeq
2x250 run Sequencer reads 250bp at a time
Run yielded poor-quality data (low diversity) Spiked with PhiX, re-run
Steps of Sequencing Sequencing Mate Pairs
Gel 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 pooled Pooled library was diluted with 0.2N NaOH and
hybridization buffer Mixture was diluted again with hybridization buffer Placed on the MiSeq for mate pair sequencing
Run yielded poor-quality data (low diversity) Sample was spiked with PhiX, re-run
Assembly First, DNA quality is charted and basic stats are
reviewed (FastQC) Use charts to find which bases to trim
Trim 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 scaffold
Results H. axyridis genome is about 300 million bp
long After trimming, we ended with…
628,908 paired end reads 4,038,064 singletons 1,454,689 mate pair reads (non-gel) 199,700 mate pair reads (gel)
Low diversity suggests transposon activity in genome
Genome full of long ‘A’ sequences
AcknowledgementsThanks to the W. Harry Feinstone Center
for Genomic Research and especially the Sutter Lab for allowing me to intern with them.
Special thanks to Dr. Shirlean Goodwin, Dr. Thomas Sutter, and Dr. Michael Dickens for their help during my time in the lab.
Disclaimer This is an informal presentation;
information taken from various print and Internet sources
Images are not mine Google, Illumina Technologies