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Yes, it depends on what you are doing. The example in the video is for a full gene knockout / replacement with a knamycin resistance gene. If the knockout is successful, mutants can be selected on media supplemented with kanamycin. Other methods may rely on specific gene disruption by inserting sequences or deleting parts of coding sequences. Best wishes.

@genomeprojects and is it necessary to put promoter also

In the video, the kanamycin resistance gene is expressed using a promoter already in the genome at the knockout site. You could probably use an existing promoter or insert one.

Hello, apologies for the later reply, I have been away. After rearranging, any colored blocks in the first, reference genome should be connected by lines to similarly colored blocks in the other genomes. The lines and colours show which regions are the same or similar between the genomes. Its hard to say without seeing your alignment, but, if the blocks are all one colour, it might indicate the compared genomes are organised very similarly?

Yes, the program can display sequences that are either long or shorter in both circular or linear forms.

Thanks, I will bear this in mind for future vids. Best wishes!

Hi, as far as I know - this was first described: Steffan Ho et al., (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene,doi.org/10.1016/0378-1119(89)90358-2. There are more recent papers including: Horton, R.M. PCR-mediated recombination and mutagenesis. Mol Biotechnol 3, 93-99 (1995). doi.org/10.1007/BF02789105.

Great! Glad you thought it was well described. Best wishes!

You're welcome! Glad you liked the vid.

Hi, I am not aware of a specific alignment tool available in SMART itself. It is possible to copy the predicted domain sequence from the SMART output and align these (and other sequences) separately. SMART also has links to InterPro, this is another site to predict protein domains. It is possible that alignment is possible through other protein domain prediction sites. What you are looking for might depend on whether you are working with a set of domain sequences that you have put together yourself (e.g. in FASTA format) - or want to compare a predicted domain sequence to others present on a sequence database. I hope this helps.

If you have annotated your genome using RAST, there are options to search using the SEED viewer. The viewer allows search for key words, or search by function etc. Finding a gene by name may be dependant on whether it has been suitably annotated rather than noted as "hypothetical" by RAST. If the SEED viewer is no help, you could BLAST search your entire genome with your gene (or similar) sequence, the BLAST search will then allow you to identify the gene coordinates within your annotation file, there you should find the gene.

@@genomeprojects ok sir. I have another doubt sir. How to identify the gene location or locus tag/ number in rast sir.

Hi, just wondering what species are you working on?

Lactobacillus iners, apparently my polished genome has just aligned to my reference genome

Yes, I heard about this. The website states: "The software remains freely available, but is no longer under active development by the original authors. There is no guarantee of support or that any emails to the authors will be answered (not that there ever was such a guarantee!)." I have a copy I downloaded and it still works fine. The program can still be downloaded (from the website) and run - it will just not be updated in the future. darlinglab.org/mauve/download.html Best regards.

Hi, There are probably several ways of doing this, here is one that works. You will need to open and make changes to the sequence/annotation in Artemis. First open Artemis with your sequence and select "save in between sessions" In Artemis, select the coding sequence/gene of interest that you want to annotate and right click. From the menu that appears, select "edit" tab, then "select feature in editor". This will open up a box showing sequences as well as gene and gene product names. The text in here can be changed. Change the gene and gene product names to whatever you want. When all gene names have been added or changed in Artemis, then save and goto file and select: Open in DNAplotter. Opening DNAplotter from Artemis will reflect the changes made. In DNAplotter, go to options and select DNAwizard, select "edit current DNA display" you can see the changes made in Artemis are now in DNAplotter. To go back to the version you made changes to, open the saved Artemis file then re-open in DNAplotter.

Glad if this was helpful to you 👍 Best regards.

Hi, it was created using screen clips from the website. These were merged in PowerPoint then saved and uploaded as a Jpeg.

Great, glad you thought the vid was useful. Best wishes from GenomePJS

Thanks for leaving such a lovely comment! I am glad you liked the video.

Hi, my apologies if you find it misleading. Thumbnail is from a screenshot taken of the RAST website, as per the contents of the video.

Usually clusters contain genes that are functionally related. Genes in cluster are also located in close proximity to one another within a genome. To identify clusters, I would predict gene function and look for cluster conservation in other / similar species. For bacterial genomes, many functionally related genes are encoded in operons and so predicting operons may also help to identify clusters.

Hi, glad the videos help. When opening a full annotation file (like .gbk) in DNAPlotter, genes are shown by default on forward and reverse strands - depending on their predicted orientations. If you go into: options; DNA wizard; Edit current display and then select: Linear (at the top left of the panel) you can then see the two tracks, forward and reverse in a linear rather than circular form. You can zoom in by pressing ctrl I. If you want to add arrow heads or tails to illustrate gene orientations, go into DNA Wizard, find the CDS using the gene coordinates and then click the boxes marked either arrow heads or arrow tails. I hope this helps to answer your question. Best wishes!

Hi, not exactly sure about the error code. Info on the web indicates this might be associated with Windows components needing an update. I would first try putting any files to align on your desktop and working from there - if they are not already. Also try only aligning a couple of genomes first (if you have a lot) in case the error relates to your memory availability for MAUVE to complete the tasks. You could also try "right click on MAUVE" and troubleshoot compatibility. Hope this helps,

@@genomeprojects Thanks a lot for your answer! I´ll try that

Hi, I have done a part 2 vid if this helps. Apologies if this vid is hard to follow. The PCR products used to create the final construct are: HA1, HA2 and Kan. HA1 is joined to Kan using primers HA1 fwd and Kan reverse. HA1 + kan is joined to HA2 using primers HA1 forward and HA2 reverse.

Hi, I just tried this myself. I first opened a GenBank (GBK) file in DNAPlotter. By default this opens up an image where the predicted CDS on either forward or reverse strands are on separate tracks (seen as concentric circles). Are you using a GenBank file - or other genome annotation file - in DNAPlotter?

an easy tutorial is on the link: home.cc.umanitoba.ca/~psgendb/tutorials/artemis/dnaplotter/dnaplotter.html#:~:text=DNAPlotter%20is%20used%20by%20Artemis%20to%20render%20maps,shows%2C%20from%20the%20outer%20circle%20and%20going%20inwards%3A

@@genomeprojects I am using a fasta file. Do I have to use a genbank file?

@@elMARABIYOCHO1989 you need to use an annotation file to see forward and reverse genes. Using a plain nucleotide sequence file, nothing has been predicted as forward or reverse yet.

@@genomeprojects What can you recommend for annotation? I used GenemarkS and then just used the Add Feature in DNAplotter to add the CDS

Not had this problem (for Artemis anyway). Could try to update or check your current Java edition / Run Time Environment is compatible with Artemis. Remove old versions of Java from system. Could also try Right click on Artemis and try troubleshoot compatability or reinstalling Artemis altogether. Hope you get it sorted.

@@genomeprojects I changed the java version and it now works. Thank you so much😀

Cool...glad you got it to work. Well done!

@@genomeprojects How do you get to have the forward and reverse strands on different tracks? I tried updating tracks and it just deletes everything. I have had to restart multiple times since it deletes everything each time I hit update tracks

One way of doing this: In DNA plotter - go into options - then into DNA WIzard, you can adjust the labels there to COG identifiers. Or, chose the linear genome configuration in DNA Wizard, zoom into the area of interest, double click on the CDS and change / add a COG label there.

Hi, think I've had a few like that. I also checked gene sequences against those in other strains or species to see if there was a difference in my sequence alone. Also had to check sequencing coverage for the region to check for assembly error caused by low coverage etc...had some gene clusters riddled with errors caused by this. Hope you get it all sorted out. BR GenomePJs

Cool, glad you liked the vid!!!

Hi, the software should be available at: sanger-pathogens.github.io/Artemis/Artemis/ Good luck with the studies!! Genome PJs

Great! Hope the vid was interesting and helped.

Hi, no problem at all. I hope it was useful.

Hi Leon, it would be best to find someone running MAUVE on Linux and ask them. I did see a discussion of the MAUVE/Linux/error topic on the Biostars website if this helps. www.biostars.org/p/159917/ Good luck and I hope you get it sorted out.

I think it might be hard to say from a draft genome sequence which contigs are truly inverted and sequencing using long reads could prove it. You might have evidence these are on the reverse as the contig mover aligns and orientates draft contigs to the order and orientation of genes in the reference. It’s difficult to say without seeing the alignment but it is possible that some genes have inverted in your draft genome - with these genes not being inverted in the reference (this does happen).

@@genomeprojects thanks your response.

Great! glad it helped.

Hi, I took this information directly from the RAST website. Go to RAST and within the login screen, select "register for a new account". You need to enter your first and last name as well as your email address into the required fields. Then select your country and choose a login name. After a RAST administrator has approved your account, RAST send you an email confirming account approval, and explaining how to login and set your password.

Hi, I added a short vid on labeling genes in DNA plotter, I hope this helps.

Cool! - if you have and useful links for that, please let me know.

Hi, these are tab-delimited files. If you open Excel, then go to: File - open, and select either your SNP, Permutation, Ortholog or Gap file. This should open them in Excel, although you may need to format the data into columns when opening Excel for some of these. Alternatively you can open these files in Notepad then copy and paste the data into a blank Excel sheet, I find this a good option.

Okay. I will try this, and let you know my experience. Thank you!

It worked! Thanks very much.

@@greaterk.oyejobi7893 Hi; to find orthologs, is this working well?

Great, I only really use RAST for annotations. RAST does have a genome browser and comparison tool also but I don't use these so much.

Hi, I am glad you found this useful.

Hi, my thoughts on this are - yes, also you would probably want to make sure the sequenced genome of your species of interest was assembled into a single chromosomal sequence first. Paraphrased from the Mauve website: “Mauve works best on closely related organisms. As genomes diverge from ancestral species their chromosomes undergo numerous rearrangements. Even after long evolutionary periods, many sequences remain but these can be present in different arrangements from one species to the next. In Mauve, the Localized Co-Linear Blocks (LCBs - colored blocks) are regions of the chromosome that appear to be conserved across the species being analyzed”. Using Mauve you could align a number of whole / similar genomes against the genome you are interested in.

@@genomeprojects thank you so much, and how would you save the new align sequence?

@@eswinipi Hi, when in Mauve, press the “file” tab and select “align with progressive Mauve” below the “sequences to align” box there is a box marked “output”. If you press on the tab with the three dots next to the output box, a new window will appear asking you where you want to save your Mauve output files to. Once you have decided where to save the output files, they will save to that location automatically once the alignment has completed. You can save your output files to wherever you want really, but I find it easier using a new folder specifically for my alignments e.g. I create a new folder and call it something like alignments on the deskstop. When you want to open a past alignment, open Mauve and press the “file” tab and then “open alignment”.

@@genomeprojects Thank you so so much! can your reference genome be in .fasta file instead? whenever I try a .gb file it keeps giving me and "exit error 3" and yes I already saw my new aligments saved on my new created folder, I was just wondering if Mauve saves those new aligments in .fasta or .gb files.

@@eswinipi Hi, if you align Fasta files, you might not have any annotations in your alignment - all that you will be able to see is the aligned nucleotide sequences (no gene names etc..). If you use a GenBank file make sure it is the full GenBank file. E.g. If exporting a genome from NCBI select “GenBank full” rather than export “GenBank” or you will not have any sequence / annotation information in the file. If you have the correct “full” GenBank files, (not sure if) exit error 3 might be to do with running out of memory to complete the alignment task, you could try aligning only a couple of genomes first just to check. I have had a look at the output files from a completed alignment, although one of the larger size files can be opened in notepad and contains genome sequences in a Fasta format, I don’t think the output files themselves are much use outside of Mauve - I have not used these for anything else myself that is. Hope this helps.

Sure, post a question and I will try and help.

Hi PubMLST, Thanks for putting this straight, this might explain why some isolates do not have sequence data when I try to export contigs via the isolate collection link. Thanks again for your advice - I love the new PubMLST look by the way and am slowly feeling more confident finding my way around your database.

Might depend what is is annotated as in your reference genome. If you can find the gene in your GenBank file this will also give you a Locus Tag that you can search for in MAUVE rather than search for gene or product. You can also find the coordinates of the gene from your annotation file and then zoom into that region in MAUVE.

@@genomeprojects Okay thank you so much! Another question, if I put in 2 genomes how do I make 1 of them the reference genome?

@@cindyoctaviana9842 Your reference genome would normally be a complete genome I.e. a single annotated chromosome assembly - not a genome in numerous contigs. When using progressive Mauve to align a genome to a reference, it is best to add the reference first - there is however an option to move the order of the genomes after the alignment has completed. On the far left of the alignments there should be something that looks like: ^ - R V You can use this to move the positions of the genomes and set which you want to use as a reference after alignment has completed.

@@genomeprojects sorry for the late reply, thank you so much for your help! it worked :)

Error in Mauve? I would check the file types you are using Genbank or FASTA etc.. as well as where you are saving the output files - in case there is a problem saving them to the location you have stated. Just a thought, I had issues once saving them to a USB.

@@genomeprojects My entire group is having problem where gb file gives an error. But when we only use fasta the program works fine

@@louismostert2650 if you are downloading the genome from NCBI - when on the page with the genome. In the "send to" drop down menu @ top of page, try selecting: complete record then select the destination as "file". Then in the drop down "format" options select GenBank (full) rather than just GenBank. Saving the file in this format has worked for me in the past.