2012-05-16T20:22:15-04:00 http://www.geneious.com/userforum/ Lussumo Vanilla http://www.geneious.com/userforum/comments.php?DiscussionID=2189 2012-03-20T12:09:18-04:00 2012-05-16T20:22:15-04:00 mejorin http://www.geneious.com/userforum/account.php?u=2451

First of all, thank you very much for providing the micro satellite plugin, which I think will eventually prove to be extremely useful for many users. I am currently testing it and have some questions related to its use: 1) all my samples have been run with the same ladder: would it be possible to set it 'a priori' for all of them, and not use the automatically check? In fact, the ladder was often not called at all or incorrectly called: and it is not clear to me how I can edit the peaks to change the ladder (I have tried deleting/creating ones, but with no avail). 2) I have noticed some annoying between samples shifts in the ladder+peaks, i.e. the ladders are not 'aligned' and therefore otherwise equivalent peaks are assigned to distinct bins. Probably this derives from the (incorrectly) called ladders? Is there a way I can align the ladders? (even scaling the X axes does not completely remove the artifactual different peaks: again, this may be related to the wrong ladder calling). Thank you, Lino

I am currently testing it and have some questions related to its use:
1) all my samples have been run with the same ladder: would it be possible to set it 'a priori' for all of them, and not use the automatically check? In fact, the ladder was often not called at all or incorrectly called: and it is not clear to me how I can edit the peaks to change the ladder (I have tried deleting/creating ones, but with no avail).
2) I have noticed some annoying between samples shifts in the ladder+peaks, i.e. the ladders are not 'aligned' and therefore otherwise equivalent peaks are assigned to distinct bins. Probably this derives from the (incorrectly) called ladders? Is there a way I can align the ladders? (even scaling the X axes does not completely remove the artifactual different peaks: again, this may be related to the wrong ladder calling).
Thank you,
Lino]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2302 2012-05-11T13:49:55-04:00 2012-05-15T22:04:59-04:00 Ebezault http://www.geneious.com/userforum/account.php?u=2130

Hi, I would like to have your feedback on how to map a large number of sequences onto a reference genome and turn these aligned regions into annotations of this genome in an automated way. In practice, I have a large batches of assembled transcriptome sequences that I would like to align/map onto the reference genomes for annotation, using Geneious (v5.6.2 // under Mac OSX). My issue here is double: 1) The reference genome assembly is scaffolded (i.e. Typical of complex genomes sequenced by NGS only). To map the gene/exon sequences onto the reference genome, I tried to use the Map_to_Reference tool in the "Align/Assemble" folder. However, this tool does not support that the "reference sequence" is constituted of such large number of "elements" (i.e. here ~8000 scaffolds). It seems that the tool is opening each element of the reference as a separate file, leading to an error message ("To many open files") when the number of elements is "too high" (even if the Operating system does not have such intrinsic limitation?!). The max-number of elements to allow the tool to run seems to be (empirically defined) somewhere between 4500 and 5000... (note: The sequence length of the elements themselves does not seems to influence this) I am wondering if this limitation could not be fixed (e.g. limit eliminated or raised by 1 order of magnitude) to allow the use of this tool on "any" genome assembly, even large non-model plant & vertebrate genomes produced by NGS which are implicitly scaffolded? Note: by splitting the reference genome into sub-groups containing fewer elements than the limit, the process seems to work... however this increases significantly the number of steps upstream and downstream the analysis itself, which is not convenient and error-prone! Alternatively, I have tried a BLAST approach, using the Sequence_Search tool, with Discontinuous_MegaBLAST after having created a local data-base with the reference genome sequence. While alignment are identified, the outcome of it does not seems to be very helpful for the following part of my purpose: - the analysis return only the aligned sequences matching the shortest one, i.e. target-sequence (the longest, here the scaffold reference-sequence, being trimmed to the size of the target-sequence), which then do not allow to "Add_ & Transfer_Annotation" to the reference sequence!? - even if in each alignment, the position (start and stop) of the target-sequence onto the reference-sequence (scaffold) is provided, I could not find a way to export this information in anyway (e.g. gff entrie format ideally)? 2) (semi-)automating the annotation process Once some alignments are obtained (i.e. "read assembled to scaffold" elements), one can turn a specific alignment into an Annotation onto the reference sequence by opening one of them, selecting the aligned sequence and use "Annotation/Add" tool then transfer it by "Annotation/copy to..." reference sequence. But this entire process seems to have to be done manually for each single alignment obtained and each aligned sequence if more than one per scaffold (including the typing of the name of the new annotations, while automatically proposing to use the name of the aligned sequence would be great). While that's totally fine for a handful of gene/sequence, that become unaffordable at a larger scale, without the possibility of batch processing these alignments into annotations. Maybe I did not find the right way to go? Or do you see any possibility to improve this? An "indirect" alternative would be to be able to generate from the alignments obtained by BLAST and/or Reference_Mapping a gff file, which could then be associated with the reference genome document as a new annotation_track. Any idea how to proceed for that too? Right now, the second point is really blocking me... I am sure others would like to do so too or might have found the way for it!? Thanks a lot for your help. Best, Etienne.

I would like to have your feedback on how to map a large number of sequences onto a reference genome and turn these aligned regions into annotations of this genome in an automated way.
In practice, I have a large batches of assembled transcriptome sequences that I would like to align/map onto the reference genomes for annotation, using Geneious (v5.6.2 // under Mac OSX).
My issue here is double:

1) The reference genome assembly is scaffolded (i.e. Typical of complex genomes sequenced by NGS only).
To map the gene/exon sequences onto the reference genome, I tried to use the Map_to_Reference tool in the "Align/Assemble" folder.
However, this tool does not support that the "reference sequence" is constituted of such large number of "elements" (i.e. here ~8000 scaffolds).
It seems that the tool is opening each element of the reference as a separate file, leading to an error message ("To many open files") when the number of elements is "too high" (even if the Operating system does not have such intrinsic limitation?!).
The max-number of elements to allow the tool to run seems to be (empirically defined) somewhere between 4500 and 5000... (note: The sequence length of the elements themselves does not seems to influence this)
I am wondering if this limitation could not be fixed (e.g. limit eliminated or raised by 1 order of magnitude) to allow the use of this tool on "any" genome assembly, even large non-model plant & vertebrate genomes produced by NGS which are implicitly scaffolded?

Note: by splitting the reference genome into sub-groups containing fewer elements than the limit, the process seems to work... however this increases significantly the number of steps upstream and downstream the analysis itself, which is not convenient and error-prone!


Alternatively, I have tried a BLAST approach, using the Sequence_Search tool, with Discontinuous_MegaBLAST after having created a local data-base with the reference genome sequence.
While alignment are identified, the outcome of it does not seems to be very helpful for the following part of my purpose:
- the analysis return only the aligned sequences matching the shortest one, i.e. target-sequence (the longest, here the scaffold reference-sequence, being trimmed to the size of the target-sequence), which then do not allow to "Add_ & Transfer_Annotation" to the reference sequence!?
- even if in each alignment, the position (start and stop) of the target-sequence onto the reference-sequence (scaffold) is provided, I could not find a way to export this information in anyway (e.g. gff entrie format ideally)?


2) (semi-)automating the annotation process
Once some alignments are obtained (i.e. "read assembled to scaffold" elements), one can turn a specific alignment into an Annotation onto the reference sequence by opening one of them, selecting the aligned sequence and use "Annotation/Add" tool then transfer it by "Annotation/copy to..." reference sequence.
But this entire process seems to have to be done manually for each single alignment obtained and each aligned sequence if more than one per scaffold (including the typing of the name of the new annotations, while automatically proposing to use the name of the aligned sequence would be great).
While that's totally fine for a handful of gene/sequence, that become unaffordable at a larger scale, without the possibility of batch processing these alignments into annotations.
Maybe I did not find the right way to go? Or do you see any possibility to improve this?

An "indirect" alternative would be to be able to generate from the alignments obtained by BLAST and/or Reference_Mapping a gff file, which could then be associated with the reference genome document as a new annotation_track.
Any idea how to proceed for that too?

Right now, the second point is really blocking me...
I am sure others would like to do so too or might have found the way for it!?

Thanks a lot for your help.
Best,


Etienne.]]> http://www.geneious.com/userforum/comments.php?DiscussionID=979 2010-04-22T14:51:23-04:00 2012-05-15T17:07:43-04:00 mfkeller http://www.geneious.com/userforum/account.php?u=582

I realize Geneious is for working with Genes, but for taxonomists/systematists who work with molecular and morphological data, it would be nice if we could do this all in one program that is like Geneious. Geneious is very user friendly. But I would like to be able to incorporate a data matrix from excel or a nexus file and be able to combine molecular and morpohological, instead of using two computers or two software programs. I know there are numerous programs like PAUP* and Mesquite, but PAUP* is no longer updated. Geneious is always working on new updates and fixing bugs, so having a plugin that could incorporate morphological data matrices would be perfect for those of us, and there are many, who work on genes and the actual organism.

http://www.geneious.com/userforum/comments.php?DiscussionID=2310 2012-05-15T12:37:24-04:00 2012-05-15T16:40:53-04:00 hcreager http://www.geneious.com/userforum/account.php?u=1520

I'm wondering if there's a way to trim contigs based on coverage. I can annotate the regions I'd like to get rid of using the "Find low/high coverage" tool in the annotate/predict menu. Is there a way to tell geneious to trim all areas labelled with the low-coverage annotation?

http://www.geneious.com/userforum/comments.php?DiscussionID=2309 2012-05-15T10:11:30-04:00 2012-05-15T16:33:48-04:00 Susanne http://www.geneious.com/userforum/account.php?u=1202

Hi, is it possible to copy the names and description of sequences from an alignment? Cheers, susanne

is it possible to copy the names and description of sequences from an alignment?
Cheers,
susanne]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2311 2012-05-15T15:53:28-04:00 2012-05-15T16:31:03-04:00 hcreager http://www.geneious.com/userforum/account.php?u=1520

I am trying to do de novo assemblies on 454 data of varying read length. I want contigs that are 100% identical across the entire contig (those that have 100% identical sites) not just across eg. 400 of 450bp. I have the assembler set to 100% minimum overlap identity and 300bp minimum overlap length. I realize that I could accomplish my objective by trimming all of my reads to 300bp and then assembling, but I want the extra base pairs from the reads that have them. Is there a way that I can assemble contigs with 100% identical sites? In other words, I want all sequences to be identical in all columns). I've been sorting my contigs by % identical sites and kicking out the appropriate sequences from those that aren't at 100%, but that's a very labor intensive process...

http://www.geneious.com/userforum/comments.php?DiscussionID=2308 2012-05-14T18:53:52-04:00 2012-05-14T19:56:55-04:00 lieftingl http://www.geneious.com/userforum/account.php?u=2237

I am using Geneious Pro 5.5.6 and want to assemble 454 and Sanger reads together. I understand that this is possible. The 454 reads are in contigs and the Sanger reads are as the original ab1 file in the same folder. To do the assembly I select all the contigs and reads, then choose Dissolve contigs and reassemble. In the resulting assembly none of the contigs contained a mixture of 454 and Sanger reads. When I did Blast2Seq (NCBI) on the contigs consisting of Sanger reads versus all the contigs of 454 reads, the Sanger contigs had up to 99% identity over a length of 270 bp suggesting that the 454 and Sanger reads should have assembled together. What am I doing wrong? Thanks

http://www.geneious.com/userforum/comments.php?DiscussionID=2307 2012-05-13T22:57:55-04:00 2012-05-14T11:35:42-04:00 wiart http://www.geneious.com/userforum/account.php?u=318

Hi, I've been using the "Find Protein Domains With InterproScan" plugin in Geneious, that work very well! I just do have a question/feature request for the plugin: when I click on one of the annotated domain, a yellow popup is displayed with many information from the InterProScan result, such as: Name: Inhibitor_I9 Type: Pfam Length: 83 Interval: 34-116 Residues: SYCCCHSHCHAYSC Database: PFAM Id: PF05922 Name: Inhibitor_I9 InterPro ID: IPR010259 InterPro NameL Proteinase inhibitor I9 InterPro Type: Domain Would it be possible to make the identifiers linkable, so that I can go to the PFAM, InterPro, ... webpages in 1 click. For now, I have to write it down, then go to the corresponding website, and then type it in. It makes the process cumbersome when I have several hundred sequences... Many thanks, and congrats for Geneious and this great plugin! Laurent

I've been using the "Find Protein Domains With InterproScan" plugin in Geneious, that work very well!

I just do have a question/feature request for the plugin:
when I click on one of the annotated domain, a yellow popup is displayed with many information from the InterProScan result, such as:


Name: Inhibitor_I9
Type: Pfam
Length: 83
Interval: 34-116
Residues: SYCCCHSHCHAYSC
Database: PFAM
Id: PF05922
Name: Inhibitor_I9
InterPro ID: IPR010259
InterPro NameL Proteinase inhibitor I9
InterPro Type: Domain

Would it be possible to make the identifiers linkable, so that I can go to the PFAM, InterPro, ... webpages in 1 click.
For now, I have to write it down, then go to the corresponding website, and then type it in.
It makes the process cumbersome when I have several hundred sequences...

Many thanks, and congrats for Geneious and this great plugin!
Laurent]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2306 2012-05-13T17:55:37-04:00 2012-05-13T19:18:43-04:00 hros001 http://www.geneious.com/userforum/account.php?u=138

I find it frustrating that the *.geneious format is not backward compatible. I could understand it if some of the embedded items were not, but for the whole thing not to be seems poor. I have tried to distribute fastq files to my class via a *.geneious file. I have a newer version of Geneious than is installed in the teaching labs. They can read the individual *.fastq files but not a *.geneious archive of them. This just causes more work and confusion. Howard

Howard]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2305 2012-05-13T16:31:06-04:00 2012-05-13T17:27:20-04:00 markholmes http://www.geneious.com/userforum/account.php?u=2520

Can you tell me how I'd conduct a search of a single sequence string, or a set (i.e. in an AnnotatedPluginDocument[]). I can see some likely methods in the api documentation but it would be really helpful if you could give me some example code. I'm needing to search very large numbers of short reads from some Illumina sequencing. Any suggestions for the fastest possible way to search would be appreciated. The find function in Geneious seems to be pretty fast compared to my own attempts in Java. Many thanks

I'm needing to search very large numbers of short reads from some Illumina sequencing. Any suggestions for the fastest possible way to search would be appreciated. The find function in Geneious seems to be pretty fast compared to my own attempts in Java.

Many thanks]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2303 2012-05-11T23:32:51-04:00 2012-05-13T16:57:28-04:00 samiam http://www.geneious.com/userforum/account.php?u=2538

I've been trying out the "Highest Quality" setting to generate consensus sequences from the assembly of fastq illumina data to a reference sequence. I've been trying figure out how the consensus is generated using "Highest Quality" and all I could find was: "When the aligned sequences contain quality information in the form of chromatograms, you can select Highest Quality to calculate a majority consensus that takes the relative residue quality into account." Am I missing an explanation somewhere else? What threshold is being used (on the top quality bases)? (0,25,75 or 100%) for calling a base? Thank you for any clarifications

I've been trying figure out how the consensus is generated using "Highest Quality" and all I could find was: "When the aligned sequences contain quality information in the form of chromatograms, you can select Highest Quality to calculate a majority consensus that takes the relative residue quality into account." Am I missing an explanation somewhere else?

What threshold is being used (on the top quality bases)? (0,25,75 or 100%) for calling a base?
Thank you for any clarifications]]> http://www.geneious.com/userforum/comments.php?DiscussionID=254 2007-12-12T05:41:47-05:00 2012-05-13T16:51:43-04:00 pharris http://www.geneious.com/userforum/account.php?u=210

Is it possible to manually add/remove a sequence from an existing alignment? For example, I often use a large reference sequence as a guide while assembling multiple sequence files from another specimen. Occasionally I have a sequence that doesn't align using the automated function, but I know where the sequence should be relative to the reference sequence and I would like to manually add this sequence to the existing alignment. Also, once I have assembled the multiple sequences in the alignment file, I would like to remove the reference sequence so that I can generate a new consensus sequence for this specimen. Any help/suggestions would be most appreciated.

http://www.geneious.com/userforum/comments.php?DiscussionID=2301 2012-05-11T13:44:36-04:00 2012-05-13T16:11:55-04:00 sbrubaker http://www.geneious.com/userforum/account.php?u=967

Hi, we just upgraded to Geneious 5.6.2 and are having errors when copying things from the server database to a local database, or from a local database to the server database. This is actually not happening on my system, but has been reported on several users systems. They did the upgrade and went through the process of backing up and converting their local data. I upgraded the Server DB. The error is either a permission denied (database) error, or an error "Failed to import 1 document to database: Failed to load additioanl XML ..."

This is actually not happening on my system, but has been reported on several users systems. They did the upgrade and went through the process of backing up and converting their local data. I upgraded the Server DB.

The error is either a permission denied (database) error, or an error "Failed to import 1 document to database: Failed to load additioanl XML ..."]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2304 2012-05-13T07:09:22-04:00 2012-05-13T16:02:46-04:00 jeremyaustin http://www.geneious.com/userforum/account.php?u=557

I've just searched NCI using Geneious using the string "organism = Gallus, AND any field = mitochondrion, AND any field = complete genome". I get no results When I go to the NCBI website and do the same search I get 69 results. Any idea why the difference?

When I go to the NCBI website and do the same search I get 69 results.
Any idea why the difference?]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2295 2012-05-08T18:03:37-04:00 2012-05-13T15:59:00-04:00 tanklao http://www.geneious.com/userforum/account.php?u=875

I wish Geneious can map protein seq to a reference DNA seq. Just like the EBI's wise2 program, it should recognize the intron and exon.

Just like the EBI's wise2 program, it should recognize the intron and exon.]]> http://www.geneious.com/userforum/comments.php?DiscussionID=490 2009-02-15T03:21:25-05:00 2012-05-11T15:11:03-04:00 dakr002 http://www.geneious.com/userforum/account.php?u=355

Hi, I wish there was the option to have every annotated CDS translated into its correct reading frame and have the resulting amino acid sequence displayed next to the DNA sequence for each feature. This could simply be implemented as an addition to the "Frame" pulldown menu in the Residues/Translation subsection (of course I do not know if it is technically that simple, but as a user that's the place where I would greatly welcome this function). Alternative (or additionally), the context menu for a CDS feature could have a command "translate this CDS", which would then add the translation of the correct reading frame next to the sequence. cheers, Daniel

I wish there was the option to have every annotated CDS translated into its correct reading frame and have the resulting amino acid sequence displayed next to the DNA sequence for each feature. This could simply be implemented as an addition to the "Frame" pulldown menu in the Residues/Translation subsection (of course I do not know if it is technically that simple, but as a user that's the place where I would greatly welcome this function).

Alternative (or additionally), the context menu for a CDS feature could have a command "translate this CDS", which would then add the translation of the correct reading frame next to the sequence.

cheers,
Daniel]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2300 2012-05-11T02:45:03-04:00 2012-05-11T02:45:03-04:00 frediezfmk http://www.geneious.com/userforum/account.php?u=2378

Hello again, I would like to submit some hundreds of barcode sequences to Genbank and wanted to ask whether this is a good idea with Geneious? I have the voucher data (collection date, collector, PCR primers, identifier etc.) annotated from the FIMS to the sequence data, there is no problem. I tried a submission with few only, but had no success. Do I have to annotate each single sequence, or how do you apply the same annotations to some hundreds of sequences. Also, do I have to manually type in necessary genbank features like start_codon position? Here are the last errors related to genbank features I got when I tried: ERROR No protein Bioseq given FEATURE: CDS: [lcl|Ex37C6:1-652] [lcl|Ex37C6: raw, dna len= 652] ERROR Missing stop codon FEATURE: CDS: [lcl|Ex37C6:1-652] [lcl|Ex37C6: raw, dna len= 652] ERROR Expected CDS product absent FEATURE: CDS: [lcl|Ex37C6:1-652] [lcl|Ex37C6: raw, dna len= 652] Unfortunately I did not find an answer to my problem so far, any advise would be great. Many thanks, Matthias

I would like to submit some hundreds of barcode sequences to Genbank and wanted to ask whether this is a good idea with Geneious? I have the voucher data (collection date, collector, PCR primers, identifier etc.) annotated from the FIMS to the sequence data, there is no problem.
I tried a submission with few only, but had no success. Do I have to annotate each single sequence, or how do you apply the same annotations to some hundreds of sequences. Also, do I have to manually type in necessary genbank features like start_codon position?


Here are the last errors related to genbank features I got when I tried:

ERROR No protein Bioseq given FEATURE: CDS: [lcl|Ex37C6:1-652] [lcl|Ex37C6: raw, dna len= 652]
ERROR Missing stop codon FEATURE: CDS: [lcl|Ex37C6:1-652] [lcl|Ex37C6: raw, dna len= 652]
ERROR Expected CDS product absent FEATURE: CDS: [lcl|Ex37C6:1-652] [lcl|Ex37C6: raw, dna len= 652]

Unfortunately I did not find an answer to my problem so far, any advise would be great.

Many thanks,

Matthias]]> http://www.geneious.com/userforum/comments.php?DiscussionID=2298 2012-05-09T03:29:55-04:00 2012-05-11T02:28:46-04:00 frediezfmk http://www.geneious.com/userforum/account.php?u=2378

Hello, my institution has 8 concurrent licenses managed via a license server that can be accessed only within the intra-net. What is the best way to make 1 license available to be used from outside? Is that possible via a VPN client installation or how could that work? It would be great if you could give me some recommendations with which I can convince our IT staff here to make this possible. Thank you & best wishes, Matthias

my institution has 8 concurrent licenses managed via a license server that can be accessed only within the intra-net. What is the best way to make 1 license available to be used from outside? Is that possible via a VPN client installation or how could that work?

It would be great if you could give me some recommendations with which I can convince our IT staff here to make this possible.

Thank you & best wishes,

Matthias]]> http://www.geneious.com/userforum/comments.php?DiscussionID=195 2007-08-23T16:19:40-04:00 2012-05-10T20:19:33-04:00 cobbler http://www.geneious.com/userforum/account.php?u=137

How do I cite Geneious in a scientific publication?

http://www.geneious.com/userforum/comments.php?DiscussionID=2299 2012-05-10T17:27:09-04:00 2012-05-10T17:49:50-04:00 minion http://www.geneious.com/userforum/account.php?u=2473

Hi, Another questions in addition to the silent restriction site one. Does Geneious 'hide' a portion of the sequence like greying it out so it doesn't affect the alignment? I'm talking more like Lasergene software where if you were cloning etc, just hide a portion of the primer and it gets greyed out. I'm finding myself having to design more primers and have a different file to see with the tag and another without. Not sure if there is something already like this, all I do is delete the portion. Thanks

Another questions in addition to the silent restriction site one. Does Geneious 'hide' a portion of the sequence like greying it out so it doesn't affect the alignment? I'm talking more like Lasergene software where if you were cloning etc, just hide a portion of the primer and it gets greyed out. I'm finding myself having to design more primers and have a different file to see with the tag and another without. Not sure if there is something already like this, all I do is delete the portion.
Thanks]]>