Transcript Gene%20Sequencing[2]

Genome
Sequencing:
Harmonia
axyridis
Isabel Risch
University of Memphis
W. Harry Feinstone Center
for Genomic Research
May 28, 2013-June 14, 2013
Ladybugs (Ladybirds)
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Tennessee state insect
Coccinella septempunctata
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Seven-Spotted Lady Beetle
Native to North America; being outcompeted by Harmonia
Harmonia axyridis
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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
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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
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Chromosomes: DNA wrapped around
histones
Human Chromosome Painting
Genome: What Is It Made Of?
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DNA (deoxyribonucleic acid)
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Called the “molecule of life”
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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
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Codes for all proteins that make cells (life) possible
Allows for the breaking of hydrogen bondsreplication
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?
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DNA and Heredity
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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)
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Mendel’s Law of Segregation
Alleles express themselves as phenotypic traits;
thus, DNA determines heredity
Genome: What Can We Do
With This Information?
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By determining the sequence of genomes, we
can…
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Compare them to other genomes
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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
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Use them for forensic science
Research deeper into genetic engineering of
plants and animals (biotechnology)
Genome Mapping
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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
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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
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Somatic diseases
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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
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Ex: sickle cell anemia is related to a mutation in the beta hemoglobin gene
DNA Sequencing: Background
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Sanger Method
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Used to determine nucleotide order in DNA
Rapid DNA sequencing
Uses modified, labeled nucleotides to stop DNA
strand elongation at specific bases
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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
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Uses fluorescent dyes to visualize all bases on one lane
DNA Sequencing: Background
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Illumina Technologies
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Next-generation sequencing
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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
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“Personal” tabletop sequencer
Capable of many of the functions of a large
sequencer
Uses fluorescence and LED light while
previous machines used lasers
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Cheaper– now many universities can
afford sequencers
DNA Extraction
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The process of separating pure genomic DNA
from the rest of the contents of cells and
tissues
Steps:
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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
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Tagmentation, amplification, etc.
 Run
on a sequencer
 Alignment and re-assembly
Genome Sequencing
Harmonia
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Why we sequenced it:
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What we used to sequence it:
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To better understand the insect and other beetles
close to 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
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May be due to transposon activity/repetitive elements
in the genome
Steps of Sequencing
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DNA Extraction
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Harmonia pupa homogenized
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Proteinase K added
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Precipitates waste
Isopropanol added
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Strips DNA of any more waste
Precipitation Solution added
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Precipitates waste from DNA
DNA Stripping Solution added
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Breaks down proteins surrounding the DNA (purifies)
Chloroform added
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Lyse cells reach DNA inside
Precipitates DNA so it can be separated from other parts of mixture
Ethanol wash
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Washes DNA to further purify (remove excess salt)
Steps of Sequencing
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Paired End Prep
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Followed Nextera XT DNA Prep Kit (Illumina, San Diego, CA)
Tagmentation
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PCR Amplification
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DNA is “amplified” in a polymerase chain reaction
Amplification: DNA is replicated many times over so the
sequencer can read it
PCR Clean-up
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DNA is fragmented and “tagged” (adapters added to DNA
ends) allows DNA to be PCR amplified
DNA is purified using AMPure Beads (unusable bits of DNA are
washed out)
Library Normalization
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Makes sure that the DNA quantities from each sample are equal
in the final pooled library
Steps of Sequencing
 Mate
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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
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selection
Used a Blue Pippin Prep machine (Sage
Sciences, Beverly, MA)
Yielded fragments 10kb-17kb
 Gel-free
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Yielded 3kb-15kb fragments
Steps of Sequencing
 Mate
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Pair Prep: Gel-Free
Tagmentation
Strand Displacement Reaction
 Polymerase
is used to fill gaps in DNA caused
by tagmentation
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AMPure Purification
 Usable
DNA binds to AMPure Beads; anything
unwanted in the solution, including small DNA
fragments, is washed away
Steps of Sequencing
 Mate
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Pair Prep: Gel-Free
Circularization
 Fragments
ligation
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are circularized with blunt-ended
Exonuclease Digestion
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remaining linear DNA is broken down,
removed from the circularized fragments
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Fragmentation of Circularized Fragments
 Circularized
DNA is sheared to smaller
fragments by sonication
Steps of Sequencing
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Mate Pair Prep: Gel-Free
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Purification of Mate Pair Fragments
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Usable DNA fragments bind to streptavidin
beads; everything else is washed away
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Usable DNA= fragments containing biotinylated
adapters
End Repair/A-Tailing
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Overhangs from DNA shearing are blunted
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3’ overhangs are removed; 5’ are filled in with
polymerase
An ‘A’ nucleotide is added to the 3’ ends
Steps of Sequencing
 Mate
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Pair Prep: Gel-Free
Adapter Ligation
 Indexing
adapters are added to the ends of
the fragments
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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
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Mate Pair Prep: Gel-Plus
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Tagmentation
Strand Displacement Reaction
AMPure Purification
Size Selection
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Used a Blue Pippin Prep machine (Sage Sciences, Beverly, MA)
Specific range of DNA fragment sizes are chosen and separated from rest of DNA
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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
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Paired Ends
Sample was diluted with hybridization buffer
and paired-end sequenced in the MiSeq
2x250 run
 Sequencer
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reads 250bp at a time
Run yielded poor-quality data (low
diversity)
 Spiked
with PhiX, re-run
Steps of Sequencing
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Sequencing Mate Pairs
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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
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Run yielded poor-quality data (low diversity)
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Sample was spiked with PhiX, re-run
Assembly
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First, DNA quality is charted and basic stats
are reviewed (FastQC)
Use charts to find which bases to trim
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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 coverage
Mate pairs provide scaffold
Results
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H. axyridis genome is about 300 million bp
long
After trimming, we ended with…
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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
Acknowledgements
Thanks 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
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Google, Illumina Technologies