1

2

Cloning and Sequencing Explorer SeriesBioinformatics

3

Stan HitomiCoordinator – Math & SciencePrincipal – Alamo SchoolSan Ramon Valley Unified School DistrictDanville, CA

Kirk BrownLead Instructor, Edward Teller Education CenterScience 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

Instructors

4

The application of information technology to molecular biology

Bioinformatics

5

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?

6

Sequence Data Analysis Tools

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)

7

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

8

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

9

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.

10

Download FinchTV

• www.geospiza.com/finchtv

11

Make project & folder and upload data to iFinch:Demo

12

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

13

Sequence Quality

14

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”

15

Sequence Quality

Q20= 732

Q20= 161

Q20= 238

17

Screen for poor quality sequence, vector, GAPDH family

18

Class Data Set

19

Sort Class Data into Folders

20

Record Data Information

21

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

22

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

23

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

24

Which GAPC Gene?

25

Break time!

26

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?

27

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

Sequence fragments

Run the CAP3 program to assemble the sequence fragments into a contiguous sequence

Contiguous sequence

Sequence fragments

Run the CAP3 program to assemble the sequence fragments into a contiguous sequence

Contiguous sequence

Sequence fragments

Run the CAP3 program to assemble the sequence fragments into a contiguous sequence

Contiguous sequence

28

CAP3 Program(ContigAssemblyProgram)

• On iFinch home page click “sequence assembly”

29

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)

30

Salvia Contig

A01

I01

C01

G01

31

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

32

BLAST search with contig

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

33

Break time!

34

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?

35

Determine Gene Structure

36

Workflow

37

BLAST Search Against Reference mRNA Database

• Blastn search with contig against plant Reference mRNA sequences database

• Change Algorithm parameters

38

Reformat BLAST results

• Reformat results in plain text format

• Save files to iFinch folder

39

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

40

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

41

Use blastx to Search Protein Database

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

42

Translate mRNA into Protein Sequence

43

Check Protein Sequence with blastp Search

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

44

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

45

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.comSupportWebinars