Cloning and Sequencing Explorer Series

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(No Transcript)
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Cloning and Sequencing Explorer
Series Bioinformatics
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Instructors

• Stan Hitomi
• Coordinator Math Science
• Principal Alamo School
• San Ramon Valley Unified School District
• Danville, CA
• Kirk Brown
• Lead Instructor, Edward Teller Education Center
• Science Chair, Tracy High School
• and Delta College, Tracy, CA
• Bio-Rad Curriculum and Training Specialists
• Sherri Andrews, Ph.D.
• sherri_andrews_at_bio-rad.com
• Essy Levy, M.Sc.
• essy_levy_at_bio-rad.com
• Leigh Brown, M.A.

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Bioinformatics

• The application of information technology to
  molecular biology

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Questions Concerning your Data

• Class Data Set
• Are our sequences high quality?
• Are my sequences similar to GAPDH?
• Are any of my sequences primarily cloning vector?
• Individual Clone Sequences
• Do my individual sequences align to give me a
  single long sequence?
• Are there discrepancies between my reads?
• Which GAPDH gene did we clone?
• Annotation of Clone Sequence
• What is the intron- exon structure/mRNA sequence
  of my clone?
• What is the protein sequence of my clone?

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Sequence data storage and analysis tools (iFinch
and Finch TV) Sequence comparison algorithm (NCBI
BLAST) Sequence Assembly (CAP3) mRNA sequence
prediction (BLAST and manual) Protein sequence
prediction (EMBL-EBI EMBOSS Transeq)
Sequence Data Analysis Tools
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Advanced Preparation

• Practice with iFinch using the guest account-
  highly recommended!
• Activate your iFinch account (2 months
  subscription)
• Download FinchTV onto lab computers
• Set up project and folder in iFinch
• Upload sequence data

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Guest iFinch Account

• http//classroom1.bio-rad.ifinch.com/Finch
• Username BR_guest
• Password guest
• Example data sets for each stage of process
• No uploading of data

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Your own iFinch account

• Each account has a unique URL
• http//Platenumber.ifinch.com/Finch
• E.g. http//A150936.ifinch.com/Finch
• Instructors Username Platenumber e.g. A150936
• Instructors Password Platenumber e.g. A150936
• Student Username Platenumber_student e.g.
  A150936_student
• Student Password Platenumber e.g. A150936
• Once activated- change your passwords!
• Active for 2 months.

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Download FinchTV

• www.geospiza.com/finchtv

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Make project folder and upload data to
iFinchDemo
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Student Activities

1. Review data quality and view sequence traces
2. Use BLAST for preliminary check on which GAPDH
   was cloned
3. Assemble sequences into a contig
4. Verify which GAPDH gene was cloned
5. Predict intron exon boundaries and generate mRNA
   sequence
6. Predict protein sequence

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Sequence Quality
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Q20 values

• The quality value of a base call is
• Q -10Log10(Perror)
• where P is the probability of an error.
• Thus if the chance that a base call is incorrect
  is 1/100, P would be 0.01 and the quality value
  would be 20 (Q20).
• Convention rates sequences by the number of
  basecalls that have quality values of 20 or
  higher- a Q20 value.
• The quality values of a sequence are calculated
  automatically by software in iFinch- a common
  program for this was developed by the University
  of Washington and is called Phred

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Sequence Quality
Q20 732
Q20 161
Q20 238
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iFinch and Sequence Quality
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Screen for poor quality sequence, vector, GAPDH
family
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Class Data Set
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Sort Class Data into Folders
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Record Data Information
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Download sequences for initial screen using BLAST

• Open Guest iFinch account
• User BR-guest, Psw guest
• Click Folders
• Click Salvia folder
• Look at data
• Go back to folder report
• Click Download folder data- save to new folder
  on hard drive
• View FASTA format in MSWord or text editor
• Upload file back to iFinch

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BLAST sequences for initial screen

• Click NCBI BLAST on iFinch homepage
• Choose nucleotide search
• Browse for downloaded salvia.fsa file to upload
• Choose Others (nr etc), Select Reference
  Genomic sequences
• Choose Plants (taxid)
• Choose Somewhat similar sequences (blastn)
• Click BLAST

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BLAST Results

• All 4 sequences were analyzed by BLAST- choose
  from pull down menu at top of page
• Mouse over top bar
• Scroll down to list of homologous sequences
• E value represents the number of equally good
  sequence matches to the query sequence that would
  be expected in a database of the same size
  containing random sequences.
• Scroll down to sequence alignments
• Query Your sequence
• Subject Database matching sequence

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Which GAPC Gene?
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• Break time!

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Questions Concerning your Data

• Class Data Set
• Are our sequences high quality?
• Are my sequences similar to GAPDH?
• Are any of my sequences primarily cloning vector?
• Individual Clone Sequences
• Do my individual sequences align to give me a
  single long sequence?
• Are there discrepancies between my reads?
• Which GAPDH gene did we clone?
• Annotation of Clone Sequence
• What is the intron- exon structure/mRNA sequence
  of my clone?
• What is the protein sequence of my clone?

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Initial Screen Result

• We have cloned Salvia GAPC gene
• Now we need to put the sequences together to make
  a contig- (contiguous sequence)
• Then correct any sequence discrepancies between
  different reads

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CAP3 Program(ContigAssemblyProgram)

• On iFinch home page click sequence assembly

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Assembly Results

• Your sequence file (your input)
• Single sequences (any seqs that could not be
  assembled)
• Contigs (save in FASTA format as .txt file)
• Assembly details (Save as landscape .txt file)

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Salvia Contig
A01
I01
C01
G01
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Check for Discrepancies

• Look through assembly file for sequence
  discrepancies
• Open chromatogram files in FinchTV
• Examine actual chromatograms and use personal
  judgment to determine which base call is correct
• Correct FinchTV file and save back to iFinch (not
  available in guest account) noting the changes in
  the revision history
• If the consensus sequence has changed, download
  folder sequences again like previously and
  reassemble with CAP3 program

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BLAST search with contig
Submit contig FASTA file for BLAST search (same
database as before- plant reference genomic
database)
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• Break time!

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Questions Concerning your Data

• Class Data Set
• Are our sequences high quality?
• Are my sequences similar to GAPDH?
• Are any of my sequences primarily cloning vector?
• Individual Clone Sequences
• Do my individual sequences align to give me a
  single long sequence?
• Are there discrepancies between my reads?
• Which GAPDH gene did we clone?
• Annotation of Clone Sequence
• What is the intron- exon structure/mRNA sequence
  of my clone?
• What is the protein sequence of my clone?

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Determine Gene Structure
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Workflow
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BLAST Search Against Reference mRNA Database

• Blastn search with contig against plant Reference
  mRNA sequences database
• Change Algorithm parameters

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Reformat BLAST results

• Reformat results in plain text format
• Save files to iFinch folder

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Save Contig File in MSWord

• Delete all paragraph marks using find and replace
  command
• Save to hard drive as .rtf file with a new
  name.
• Color contig sequence with exons as determined
  from BLAST results
• Put exons together in a first draft of the mRNA
  sequence and save to iFinch folder
• Submit draft mRNA sequence to blastn against
  plant reference mRNA database

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BLAST search with derived mRNA sequence

• Correct intron-exon boundaries (use Arabidopsis
  mRNA as model)
• Resubmit to BLAST
• Reiterate if necessary until no indels are
  evident and you are satisfied with a final mRNA
  sequence
• Save to iFinch folder

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Use blastx to Search Protein Database

• Blastx converts nucleic acid sequence to amino
  acid sequence and searches protein database.

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Translate mRNA into Protein Sequence
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Check Protein Sequence with blastp Search

• Ensure translation is in correct frame
• Save to iFinch folder

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Congratulations!

• You have cloned, sequenced and annotated a novel
  gene.
• You could now submit this to GenBank.
• Data from additional samples would strengthen the
  data- for example- assemble sequences from the
  same gene from different student teams
• Download data from iFinch if you wish to keep it
  for the long term

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Webinars

• Enzyme Kinetics A Biofuels Case Study
• Real-Time PCR What You Need To Know and Why You
  Should Teach It!
• Proteins Where DNA Takes on Form and Function
• From plants to sequence a six week college
  biology lab course
• From singleplex to multiplex making the most out
  of your realtime experiments
• explorer.bio-rad.com?Support?Webinars