Post on 17-Jan-2018
description
ATGTTCTATCCCATTCATTTTGACGTTATTGTTGTTGGAGGAGGTCATGCTGGGACAGAAGCAGCTTTGGCTTCTGCTAGGATGCAATGTAATACGCTTTTGATTACTCATAATATTGATACTCTTGGACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGAAGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGATGAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCAAAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTTTCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCTTGTTATATTACCCATACTAATAACAAAACACATGAAATAGTTAGATCAAATTTATATCAGAGTCCTATGTATACAGGTTTAATAAAAGGAATAGCACCTCGGTATTGTCCATCTATAGAAGACAAAATAACTCGTTTTTCAGATCGTAATGCTCATCAAATTTTTTTAGAACCTGAAGGTTTGACAACACCTGAAGTATATCTTAATGGTATTTCTACTAGCTTACCTTTTTGTGTACAAATGCAAATGATTAAATCAATTCAGGGATTAGAGAATGCTTGTATAATTAGGCCAGGTTATGCTATTGAATACGATTTTTTTGATCCTCGTGATTTAAAATTAACATTAGAGAGTAAAATTATTTCTGGATTATTTTTTTCTGGTCAAATTAATGGTACTACTGGTTATGAAGAAGCAGCTGCCCAAGGATTATTGGCCGGAATTAATGCAGCTAGGTTTTCCAAAAATAAAGAAGGATGGTACCCTAGAAGAGATCAGGCATATTTAGGGGTGCTTGTAGATGATCTCTGTACACATGGAACAGAAGAACCTTATCGTATGTTTACTTCGCGTGCTGAATATCGTCTGTCTTTACGTGAGGATAATGCTGATTTAAGATTAACTGAAATCGCGCGACAGTTAGGTTTGATAGATGAGTCACGGTGGAAAGCTTTTTGTTGCAAAAAAGAAAACATTGAAAAAGAACGTCAGAGATTACGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAACACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAAGTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAATAAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTATAATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGACAAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTATCACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTAATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGGGTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAATAATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTATGTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAATAGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAAATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGTGTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGTGTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTCTATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACAACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTTGTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAATATTTTTTGAAAGGTGGTTTATGTCAGGAATCAAAGGCACTTCCCAAGAATATATTGGACATCATTTATATCATCTGCAATTTGATTTAAGTACTTTTTCGTTAGTGAGCTCGGAGAATACTTCTTCATTCTGGGTATTAAATGTAGATTCGATGTTTTTTTCAATATTATTGGCTACTTTATTTTTATTAATTTTCGGTCGTTTGGCTACAGTGGCAACTTATGCGGTTCCCACAAAACTGCAAGTGTTTATTGAGTTAGTAATATTGTTTATTGATAGCAATGTAAAAGATATGTTTCATGGTAAAAATAAACTAATTGCGCCATTATCTATGACGGTTTTTGTTTGGATTTTTTTAATGAATACCATGGATTTATTTCCCATAGATTTATTTCCTGCTATAGCTAAATTGTTAGGATTACCTGCTTTACGTGTTGTGCCGTCTGCCGATGTGAATATCACTTCTTCGCTAGCTTTAAATGTATTTGTACTTGTTATGTACTACAATATTTACGTTAATGGCGTTCATGGTTTTATTAAAGGACTGATGTATCATCCATTTAATCATCCAACATGTATTCCTATTAATTTTATTATTGAAGTTGTTAGTTTGTTATCTAAACCAGTATCACTTAGTCTTAGATTATTTGGCAATATGTATTCTGGTGAGTTGATTTTTATTTTGATATCTGGTTTATTACCATGGTGGGGACAATGGGTTTTAAATTTACCATGGGCTATTTTTCATATTTTAGTCGTTACATTACAGGCTTTTATTTTTATGGTTTTAACGGTGATTTATTTATCTACAGCCCATGACTCCTGTTAAAATGAATCATGTAATACAGAAGATTGCAGAGAGGTTATTATGGAACATTTAAATTTTGATATGTTATATATTGCTGCAGCAATAATGATGGGATTAGCAGCAATTGGAGCGGCTATCGGTATTGGCATCTTAGGTAGTAAATTTTTAGAGGGGGCTGCACGTCAACCAGATCTCATTCCGATCCTTCGAACTCAGTTTTTTATTGTTATGGGATTGGTTGATGCAATACCAATGATTACTGTAGGTCTTGGTTTGTATGTGATGTTTTCTGCGGTTTAACAATGAAAATTAATTGCATGTATTTATTATATTGATTATTAAATATCAGGATTTGCGCTGTGAATCTTAATGCAACAATATTAGGTCAAACTATTTCATTTGTTTTGTTTGTTTGGTTTTGTATGAAGTATGTGTGGTATCCATTTATATCTATTATTGAGAAACGCCAAAAAGAAATTTCTGATAATTTAGTTTCTGCTACTCATGCCAAAACAGAATCTGAGCGTGTCAATGCTGAAGCTTTGCTTTGTTTGAGACAGGCTCGGGTCAAAGCTCAAGAAATTATAAAACAAGCAAATAAATGTAAAATGCAAATAATTAATGAAGCTAAACATGAAGCTGAAAAGGAGCAAAGTAGAATTTTATCTCAAGCGCGAGAACAGATTATTTATGAAAGAAAACGTGTTACTGACGAATTAAGAAAGCAAATTAGCGAACTTGTAATTGAGGGTACAGAGAAAGTTATAGAACATTCTATAAATGAAATGATTGATATAGATCTTTTAAATAATATTATTAATACGTTGTCATATAAGGATTAGATGTCTAGTATGCTTGTGGTTGCGCGTACGTATGCTCAAGCTATATTTGATATAGCTGTAGAACAGAAAAATATAAACAAGTGGAAATCAGTTCTTGATTTATTTTCTGAGATTAGTCTAAATAGACTAGTACAATCTTTATTTTTTAGATGTTTAGAACCAAAAAGATTGTCAGATATATTTATTGCTATTTGTGAAGATTACCAAAAGAAACAAGTTGATACCTTCAGTAAAAATATAATCTATATTATGGCGGAAAATAATCGTTTATTATTATTACCAATTGTATTTAAAGAGTTTACTTATTTATGTTCTATATATGTTCATACTGTAGAAATAGAAATTATCTCTGCTTGGCCTTTGAAGTATAATCAGCTGAAAAAAATTACTGATATAATGGCTAAACGTTTATCTAAAACAGTGAATCCAGTACACAAAGTAGATAAAGATATATTGGCTGGTGTAATTATTCGTATTGGAGATACTGTGATTGATGGAAGTATACGTGGACGTATTTTTCGCTTAAATCACGTACTACAATCTTAATATTTTAACAGTAGTTAAAAGGTATAAACAAAAGATTATGCAATTAAATTCAAATGAAATTTCAGATAATGGAGAAGTTATTATTAATGATTTAAAGTTATTTTATAATAAAGCTAGACAAACTAAAATTACAGAAGAACTTACAGAAATTGTTTCAGGAGCTTCTGTAATATAAACTTAAAAGATAGTTAGAGGTATAATTAATATGAGTTCTGGAAAAATTGTCCAGGTTATTGGAGCGGTGGTTGATGTTGCGTTCAATCAAGATGTGGTACCGACTGTATACCATGCACTTGAGGTGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAACACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAAGTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAATAAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTATAATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGACAAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTATCACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTAATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGGGTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAATAATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTATGTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAATAGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAAATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGTGTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGTGTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTCTATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACAACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTTGTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAATATTTTTTGAAAGGTGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGAAGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGATGAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCAAAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTTTCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCT
DNA sequencingWhat, When, Why?How?Who and Where?
DNA detective
?
DNA extraction PCR
Gel electrophoresis
Insect identificatio
n
ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCA
DNA sequencingACAGATGTCTT
GTAATCCGGCC
GTTGGTGGCAT
AGGGAAAGGAC
ATTTAG
Bioinformatics
WHAT types of biological insights can DNA sequence data can provide?
WHEN and WHY would we want to obtain this detailed information?
HOW is DNA sequencing actually performed?
WHO conducts DNA sequencing, and WHERE?
The elegant idea behind DNA sequencing
Technology changes quickly, but for many years we’ve on Sanger’s cool trick.
Fred Sanger
In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators. This method is still in use today.
What is the basis of Sanger’s method?
Shared with Walter Gilbert and Paul Berg
Fascinating. Now how do we actually sequence ??
DNA Sequencing:
Step 1: Purify template (the DNA to be sequenced)
Amplified DNA+ used Taq+ extra primer and dNTPs+ salts from PCR buffer, etc….
On to sequencing….
clean PCR product + water
PCR clean-up
Step 2: Set up cycle sequencing reaction
NUCLEOTIDES
DIDEOXYNUCLEOTIDES
TEMPLATE DNA (e.g., YOUR PCR PRODUCT)
DNA PRIMER
Today, nearly all sequencing is Fluorescence-based, and uses chain terminators that are labeled with different dyes.
The dyes are “spectrally distinct,” and each has a different emission wavelength.
denaturation
Primer annealing
Product extension
Reaction steps seems a lot like PCR, but not quite…
Step 3: Perform cycle sequencing in PCR machine
1. Purify PCR product
2. Set up sequencing reaction
3. Perform cycle
sequencing
4. Resolve sequence fragments
5. Read order of terminators (DNA
sequence)
In sum: From PCR to sequence data
Quality of sequence data may vary, depending on:
• Purity and concentration of template DNA • Presence of extra PCR bands (artifacts)• Quality of dye-terminators, electrophoresis matrix, and other reagents
Ideally, look at chromatograms and convince yourself that base calls are robust.
DNA sequence assembly: Combining sequence reads to build the entire sequence of the template DNA
PCR PRODUCT of 1,200 bp
Sequence read #1
Sequence read #2
Sequence read #3
Sequence “reads” typically range from 500-800 bp. Often the total DNA fragment is much longer.
Regions of overlap among sequence reads:
• Let us piece together the linear “puzzle” of the template DNA sequence.
• Often confirm base calls and improve overall add data quality.