Exercises for the Basics tutorial

Go to parent CLC Bio Main Workbench

Genbank searches

Search for the longest human BRCA1 mRNA sequence.
Perform a Search on NCBI: Error creating thumbnail: Unable to save thumbnail to destination Twice click the header of the Length column in the results table to order the sequences according to length in descending order. Error creating thumbnail: Unable to save thumbnail to destination Download and Save the sequence.

How many times was the record updated ?

Go to the Genbank record view and look at VERSION section. Each time a record is updated, it keeps the same accession number but the version number is increased by one. In the "VERSION" section of the record you see that the version number is NM_000059.3 The ".3" means that this is the third version of this record so it has been twice updated. Error creating thumbnail: Unable to save thumbnail to destination

Select and save the CDS of BRCA1 in a separate file.
Go back to the linear view. In the Side Panel, go to the "Annotation type" settings and remove all types except "CDS". In the Navigation Area, right click the CDS annotation and select to open the annotation in a new view: Error creating thumbnail: Unable to save thumbnail to destination Save the sequence.

What is the UniSTS ID of the longest STS in BRCA1 CDS ?
STS stands for Sequence Tagged Site, these are short (200-500 nt) sequences that are known to occur only once in the genome and for which the genomic location is known. The UniSTS database contains the sequences of the primers that can be used to selectively amplify them. You can use them to screen a library of uncharacterized clones for clones that contain a specific region of the genome. If you find pairs of overlapping clones you can use them to construct contigs (STS are unique so two clones that contain the same STS must overlap). They are also used to map mutations (deletions or insertions , not point mutations) in unique regions of the genome. PCR will generate different product sizes for mutations and as such they are used to trace mutations through families.
In the Side Panel, go to the "Annotation type" settings and select "STS". Choose Fit width to see an overview of all STS. In the Navigation Area, hover your mouse over the longest STS to visualize a box containing the UniSTS ID. Error creating thumbnail: Unable to save thumbnail to destination

How many introns does the BRCA1 gene have ?
For this we need the genomic sequence instead of the mRNA sequence. So repeat the Genbank search but search for a genomic sequence that contains the complete CDS. Error creating thumbnail: Unable to save thumbnail to destination Annotate mRNA and zoom in to a level that allows you to count the introns. There are 22 introns in this gene.

Translate the BRCA1 CDS to obtain the protein sequence ?
Right click the CDS and select "Translate CDS/ORF" Error creating thumbnail: Unable to save thumbnail to destination Annotate mRNA and zoom in to a level that allows you to count the introns. There are 22 introns in this gene.

UniProt searches

Download the protein sequence of BRCA1 from UniProt ?
Perform a UniProt search Error creating thumbnail: Unable to save thumbnail to destination Open the BRCA1 protein and save.

Pairwise alignment

Align the two protein sequences to see if they correspond.

Go to Toolbox - Alignments and Trees - Create Alignment Select the two proteins: BRCA1 Protein and translation of BRCA1 CDS Use the following parameters for the alignment: Error creating thumbnail: Unable to save thumbnail to destination Deselect all Alignment Info in the Side Panel and zoom out to see the alignment on a single page. Error creating thumbnail: Unable to save thumbnail to destination You can see that the UniProt protein is a part of the translation of the Genbank CDS.

BLAST searches

Using the UniProt sequence, perform a stringent BLAST search on SwissProt to find homologs of BRCA1.
Go to Toolbox - BLAST - BLAST at NCBI Select the BRCA1 Protein sequence as an input Perform a blastp search on swissprot. We want a stringent search, so we will set a low Expect threshold: Error creating thumbnail: Unable to save thumbnail to destination Many potential homologs are found: Error creating thumbnail: Unable to save thumbnail to destination

Download the sequences that were found by the BLAST search.
Go to Toolbox - General Sequence Analysis - Extract Sequences Select the BLAST results file as an input file Extract to a new sequence list Error creating thumbnail: Unable to save thumbnail to destination Note that only the parts that are similar to human BRCA1 are saved not the full sequences !.

What is the function of the best non-human hit ?
Switch to the BLAST table view of the BLAST results. Error creating thumbnail: Unable to save thumbnail to destination Double click the second hit (in dark grey on the figure) to open the sequence. Switch to the Genbank view of the file and search for the COMMENTS section Error creating thumbnail: Unable to save thumbnail to destination

Pairwise alignment: dot plot

Create a dot plot of the BRCA1 gene and the BRCA1 mRNA.

Dot plots provide a graphical way of detecting similarity. Go to Toolbox - General Sequence analysis - Create Dot Plot Set the word size to 20. In the Side Panel of the resulting dot plot, increase the stringency of the coloring Error creating thumbnail: Unable to save thumbnail to destination Search for blue diagonal lines: they correspond to regions that are the same in the mRNA and the gene sequence, i.e. the exons. In the longest exon you see a number of internal repeats (visible as lines parallel to the diagonal of the exon Error creating thumbnail: Unable to save thumbnail to destination

Searching for annotations based on their sequence

We will use an annotated vector sequence to annotate unannotated vector sequences. The Workbench comes with a list of vector sequences, some of them annotated some of them not. In the "Example Data" folder, expand the "Cloning vector library". As you can see it contains many vector sequences but when you open and cicularize the first vector: "M13mp8_pUC8" or vector "pAT153" you can see that they do not contain any annotations.
When you open and circularize "PCS19" on the other hand, you see that it is well annotated.

To get an idea about the number of unannotated vectors, search for all vectors that contain an ampicillin resistance gene.
Go to Edit - Local Search to open the word search function in the Workbench Search for all sequences with an annotation that starts with the word "ampicillin": Error creating thumbnail: Unable to save thumbnail to destination You can see that he can only find 3 vectors that contain an ampicillin resistance gene, which is far below the number that you'd expect.

Extract the annotations from PCS19.
For this you first need to install the "Extract annotations" plugin if you haven't done so already: Go to Help - Plugins and Resources Click "Download Plug-ins" Select the "Extract annotations" plugin and click "Download and install"
After installing the plugin you can use it: Go to Toolbox - General Sequence Analysis - Extract Annotations Select the pCS19 vector and click "Next" Set the parameters as follows and click "Next": Error creating thumbnail: Unable to save thumbnail to destination Type: All We want to extract all annotations (this is not entirely true: we don't want conflicts or old sequences for instance but it is impossible to select multiple types from the drop down list so we take all). Only annotation types that are found in the selected sequence(s) are shown in the list. Flanking residues: 0 We do not want to select extra nucleotides that are no part of the annotation but that lie immediately upstream or downstream of the annotation. Select to open the results This generates a sequence list containing all extracted annotations from pCS19. Save the list and export as a fasta file.

Tranform the list of extracted annotations into a motif list.
Go to Toolbox - General Sequence Analysis - Create Motif List Click "Import From Fasta File" and browse to the file with the extracted annotations Error creating thumbnail: Unable to save thumbnail to destination Delete the annotations that you do not need like "genomic_DNA" and "Misc._feature" and one of the two "Tet" annotations by clicking "Delete" Save the motif list

Please note that the Workbench comes with a list of motifs for annotations. You can find them in the "Example Motifs" file in the "Enzyme lists" folder in "Cloning" section of the "Example Data". When you open this file you see that it contains among others motifs for the start codon, the hexaHis tag, primers for various promoters...

Search the extracted annotations in the following unannotated vector sequences: M13mp8_pUC18 pAT153 pBR325 pBR327par

Go to Toolbox - General Sequence Analysis - Motif Search Select the unannotated vector sequences in the "Cloning vector library" folder and click "Next": Select the parameters as follows and click "Next": Error creating thumbnail: Unable to save thumbnail to destination Use motif list We want to use the motif list that we created based on the extracted annotations Accuracy: 100 You can adjust the percentage identity (percentage identical residues) between the motif and the match on the sequence. If you use a list of motifs, the accuracy applies only to the simple motifs in the list. We only want to find perfect matches to the motif so we the accuracy to 100% Include negative strand We also want to find occurrences of the annotations on the reverse strand. Select to open the results and click "Finish" If annotations are found in the unannotated vector sequences they will be added. For instance, circularize pAT153 and you will see that both Tet and Amp have been annotated on the sequence.

The Workbench contains by default a list of vector sequences but this list is not complete and some vectors are not annotated. Since adding and annotating vector sequences to the Workbench is a cumbersome task, you can download an extensive list of annotated vector sequences, compiled by BITS. You can simply import this list into a folder in your Workbench.