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Cloning and Sequencing Explorer Series Bioinformatics

Cloning and Sequencing Explorer Series Bioinformatics. 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

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Cloning and Sequencing Explorer Series Bioinformatics

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  1. Cloning and Sequencing Explorer Series Bioinformatics

  2. 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@bio-rad.com Essy Levy, M.Sc. essy_levy@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

  3. Bioinformatics The application of information technology to molecular biology

  4. 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?

  5. 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

  6. 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

  7. 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

  8. Your own iFinch account Each account has a unique URL: http://Platenumber.ifinch.com/Finch E.g. http://A150936.ifinch.com/Finch Instructor’s Username: Platenumber e.g. A150936 Instructor’s 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.

  9. Download FinchTV • www.geospiza.com/finchtv

  10. Make project & folder and upload data to iFinch:Demo

  11. Student Activities • Review data quality and view sequence traces • Use BLAST for preliminary check on which GAPDH was cloned • Assemble sequences into a contig • Verify which GAPDH gene was cloned • Predict intron exon boundaries and generate mRNA sequence • Predict protein sequence

  12. Sequence Quality

  13. 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 (Q=20). 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”

  14. Sequence Quality Q20= 732 Q20= 161 Q20= 238

  15. Screen for poor quality sequence, vector, GAPDH family

  16. Class Data Set

  17. Sort Class Data into Folders

  18. Record Data Information

  19. 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

  20. 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

  21. 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

  22. Which GAPC Gene?

  23. Break time!

  24. 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?

  25. 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

  26. CAP3 Program(ContigAssemblyProgram) • On iFinch home page click “sequence assembly”

  27. 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)

  28. Salvia Contig A01 I01 C01 G01

  29. 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

  30. BLAST search with contig Submit contig FASTA file for BLAST search (same database as before- plant reference genomic database)

  31. Break time!

  32. 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?

  33. Determine Gene Structure

  34. Workflow

  35. BLAST Search Against Reference mRNA Database • Blastn search with contig against plant Reference mRNA sequences database • Change Algorithm parameters

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

  37. 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

  38. 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

  39. Use blastx to Search Protein Database • Blastx converts nucleic acid sequence to amino acid sequence and searches protein database.

  40. Translate mRNA into Protein Sequence

  41. Check Protein Sequence with blastp Search • Ensure translation is in correct frame • Save to iFinch folder

  42. 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

  43. 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

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