SeqMonk: Visualisation and analysis for large mapped data sets - Page 15

ctstackh · 12-17-2014, 11:00 AM

Quote:

Originally Posted by simonandrews

We have just released seqmonk v0.29.0 onto the project web site.

This release adds a bunch of new features. Many of these are improvements to the chromosome view, specifically targeted at studies with large numbers of samples (we're getting a lot of single cell datasets in these days). You can now display your quantitated data in some new ways, and can also look at the variability in the quantitations shown when you're displaying a replicate set.

We've also added some new probe generation and quantitation options which have proved to be useful for projects we've worked on recently.

A related release which prompted some of the new features is that we have also put up the documentation for our methylation analysis course. We'll be running this fairly regularly starting in the new year, but all of the material for the course is available for anyone who wants to look. The course isn't solely focussed on seqmonk for the visualisation and analysis, but this does make up the majority of the practicals, so anyone wanting to use seqmonk to look at methylation data might want to take a look at the material we've put up.

Simon,

I just wanted to let you know that I started using version 29 today, but I've had to force quit 3 times now because the program becomes non responsive. The issues begin after I generate an annotated probe report then try to use the report to navigate to regions of interest by double clicking them in the report. The program then freezes.

OSX 10.8.5 16GB RAM i7 core ( the program uses 10GB RAM)

kentawan · 01-26-2015, 07:11 PM

Quote:

Originally Posted by ctstackh

Simon,

I just wanted to let you know that I started using version 29 today, but I've had to force quit 3 times now because the program becomes non responsive. The issues begin after I generate an annotated probe report then try to use the report to navigate to regions of interest by double clicking them in the report. The program then freezes.

OSX 10.8.5 16GB RAM i7 core ( the program uses 10GB RAM)

Hi,

How many probes you have in your report?

kentawan · 01-26-2015, 07:43 PM

Hi Simon or anyone else,

Is it possible to edit the chromosome names in SeqMonk? I started my project using genomes downloaded from Ensembl FTP. As I proceed I realized that I needed data from UCSC Genome Browser (CpG Island estimated position to be exact, which is an annotation track). I realised that the genome labelling is different, hence I want to edit them. Any way of doing it?

regards

Ziyi

jdis · 02-02-2015, 02:09 PM

Hi Simon and other SeqMonk developers,

Thanks so much for this powerful and comprehensive set of tools!

I'm using the bisulphite feature methylation pipeline for an RRBS dataset. Re: setting the minimum level of observation required to include a base in the methylation calculation, the pipeline help page recommendation is :
"this value should be a fair reflection of the overall depth of coverage in your data"

Backing up a bit, can we use SeqMonk to find the overall depth of coverage for each CpG in the data? This is a stat that I would like to have anyway, as a measure of the quality of each RRBS library that I'm comparing. Initially I thought of creating a probe set containing all CpG dinucleotides, quantitating over these and generating a coverage histogram, but I'm not sure whether this is an option.

Any help would be greatly appreciated.

Thank you!
jd

simonandrews · 04-23-2015, 07:14 AM

I've just released seqmonk v0.30.0 on the project web site.

This version adds an optional link between seqmonk and a local R installation to allow us to easily run some R based analyses seamlessly from within SeqMonk. Initially the applications we've implemented using this link are DESeq2 and EdgeR for RNA-Seq analysis and logistic regression for replicated bisulphite sequencing analysis. We're open to suggestions for what other packages or tests should be implemented so please shout out if there's anything you think would be particularly useful.

mfahim · 04-24-2015, 07:35 PM

Hi Simon.
introducing RNAseq quantitation pipeline to seqmonk is great. Are you planning to add a pipeline for smallRNA soon?. If not how do I define probe for small RNAs.

Thanks
Fahim

simonandrews · 04-25-2015, 01:44 PM

The RNA-Seq quantitation has been in for a while, but we now make it easy to link out to DESeq and EdgeR without having to leave the program.

For smallRNAs you could still use the RNA-Seq pipeline but just change the feature type you want to quantitate to the appropriate small RNA category (miRNA for example). Given that small RNAs aren't generally spliced though you could just use a normal read count quantitation too.

There's also the small RNA QC plot which is a really useful way to start looking at your small RNA data.

mfahim · 04-26-2015, 07:34 PM

Thanks for the tip off.. Howerver, I am a bit worried about the cummulative distribution graph after percentile re-quantitation and match distribution quantitation. here is the snapshot.

simonandrews · 04-27-2015, 12:50 AM

Quote:

Originally Posted by mfahim

Thanks for the tip off.. Howerver, I am a bit worried about the cummulative distribution graph after percentile re-quantitation and match distribution quantitation. here is the snapshot.

I can't see anything attached...

mfahim · 04-27-2015, 02:31 AM

Oh. previous snapshot was too big perhaps..here is another one. Cheers

simonandrews · 04-27-2015, 03:02 AM

Is this RNA-Seq or small RNA? What is the quantitation? Did you do the corresponding QC plot for the data type you're using - if so, what did that look like? You said you did two levels of additional normalisation, were they obviously needed? Did the distribution change much after applying them?

You seem to have a very large proportion of unmeasured probes which seems suspicious. Does the raw data look the way you'd expect?

mfahim · 04-27-2015, 03:19 AM

1. is this RNA-Seq or small RNA?
Ans.Yes, this is smallRNA seq.

2. What is the quantitation?
Ans. As you mentioned previously, I applied the normal RNAseq Quantitation pipeline.

3. Did you do the corresponding QC plot for the data type you're using - if Ans. so, what did that look like?

4. You said you did two levels of additional normalisation, were they obviously needed? Did the distribution change much after applying them?

Ans. Yes, as per your youtube instruction I went to check the plot and after application of the two normalization levels. This is how it appears (the shared picture), improved as compared to the previous plot but obviously still bad.

5. You seem to have a very large proportion of unmeasured probes which seems suspicious. Does the raw data look the way you'd expect?
The QC plot was having some error, I have sent the report.
I have no idea what is happening here.

mfahim · 04-29-2015, 10:19 PM

Hi Simon,
Like many others on this forum, I am a big fan of SeqMonk.
Here is a suggestion for next version (or may be I dont understand SeqMonk).
Yes, we can make customized Annotation Tracks BUT what if I have a specific list of genes involved in a specific process then how do I make annotation track.

I guess there is room for improvement here, an option may be included where I can upload a list of genes with a variety of fuctions (names or TAIRids) and then make a customized Annotation Track. If this is added to the SeqMonk next version, that will greatly help my analysis.

If there is already one then I am sorry for oversight.

I am working with flowering time in Arabidopsis and there are several pathways involved such as Vernalization pathway, Photoperiod Pathway, Ambient Temperature Signalling Pathway, Autonomous Pathway and some Biochemical Pathways, furthermore new genes are discovered on daily basis that add to the flowering time and the TAIR database is not regularly updated.

Your kind insight will be highly appreciated.

Best Regards
Fahim

simonandrews · 04-30-2015, 12:04 AM

Quote:

Originally Posted by mfahim

Hi Simon,
Like many others on this forum, I am a big fan of SeqMonk.
Here is a suggestion for next version (or may be I dont understand SeqMonk).
Yes, we can make customized Annotation Tracks BUT what if I have a specific list of genes involved in a specific process then how do I make annotation track.

You can already make a track out of a subset of an existing track. In your case if you have a list of gene names then you could do:

Edit > Find named features

Paste your list of names into the box and select 'gene' as the features to search. It will then match your gene names against the names of the gene features and give you a list of hits. Once you have this you can press "Save All as Annotation Track" to turn the list of hits into a new track which you can then use for your downstream analysis.

Does that do what you want?

mfahim · 04-30-2015, 12:33 AM

for the Genelist..
That is exactly what I was looking for. Perfect.

More questions next week...

Cheers
Fahim

mathew · 06-22-2015, 06:13 PM

I am using Seqmonk 30. I have ran Chip analysis and has annotations like CGI, TSS, mRNA, Promoter etec. I want to export coordinates with all the annotations in one file Is it possible how to get around.

Thanks

simonandrews · 06-23-2015, 12:56 AM

Quote:

Originally Posted by mathew

I am using Seqmonk 30. I have ran Chip analysis and has annotations like CGI, TSS, mRNA, Promoter etec. I want to export coordinates with all the annotations in one file Is it possible how to get around.

If you want the raw annotations which seqmonk loads then you can just go to the Genomes folder where you'll find a set of .dat files which are EMBL format sequence header files which contain all of the positions of the annotations you see when you load a core genome.

For individual tracks you could make probes over the features using the feature probe generator and then create an annotated probe report to get a simpler tab-delimited output with coordinates in. The only thing you wouldn't get by doing it this way would be the positions of any sub-features (ie exons within transcripts).

mathew · 06-23-2015, 05:51 AM

.dat file will give the genome coordinates of that particular gene. If I create a report that will be for one annotation i.e either TSS 2000(say) bp up down / mRNA/ CGI / intergenic and so on. What I want to do is to call peaks and annotate each peak with any region whether it is in enhancer, promoter, CGI, mRNA assigning all criteria in one output file.
So I am looking for a file outlined below-

chr1 100 1000 geneXX cgi 2000bp enhancer Tss 1000 bp RPKM 1 RPKM2 Diff

simonandrews · 07-01-2015, 08:23 AM

We've just released seqmonk v0.31.0 onto the project web site. We've added some new options for analysing RNA-Seq and BS-Seq data and have tidied up the user interface for cases where you have long names for your data stores or probe lists.

http://www.bioinformatics.babraham.a...jects/seqmonk/

simonandrews · 09-09-2015, 08:21 AM

SeqMonk v0.32.1 has just been released. This fixes a bug which meant that on some systems the dialog which showed the progress of R scripts could hang. We've also taken the opportunity to improve the reporting of R crashes so that the full R log is now also sent alongside the crash report, which should help to sort out any remaining R issues.

We also improved the line graph, adding the ability to click on a line to see which probe it came from, and double click on it to go to that probe in the chromosome view.

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02-02-2015, 02:09 PM	#284
jdis Junior Member Location: boston, ma, usa Join Date: Jan 2015 Posts: 1	Quantitating RRBS coverage with SeqMonk Hi Simon and other SeqMonk developers, Thanks so much for this powerful and comprehensive set of tools! I'm using the bisulphite feature methylation pipeline for an RRBS dataset. Re: setting the minimum level of observation required to include a base in the methylation calculation, the pipeline help page recommendation is : "this value should be a fair reflection of the overall depth of coverage in your data" Backing up a bit, can we use SeqMonk to find the overall depth of coverage for each CpG in the data? This is a stat that I would like to have anyway, as a measure of the quality of each RRBS library that I'm comparing. Initially I thought of creating a probe set containing all CpG dinucleotides, quantitating over these and generating a coverage histogram, but I'm not sure whether this is an option. Any help would be greatly appreciated. Thank you! jd

04-24-2015, 07:35 PM	#286
mfahim Member Location: Pakistan Join Date: Apr 2015 Posts: 11	smallRNA Analysis Pipeline Hi Simon. introducing RNAseq quantitation pipeline to seqmonk is great. Are you planning to add a pipeline for smallRNA soon?. If not how do I define probe for small RNAs. Thanks Fahim

04-26-2015, 07:34 PM	#288
mfahim Member Location: Pakistan Join Date: Apr 2015 Posts: 11	Thanks Simon---However Thanks for the tip off.. Howerver, I am a bit worried about the cummulative distribution graph after percentile re-quantitation and match distribution quantitation. here is the snapshot.

04-29-2015, 10:19 PM	#293
mfahim Member Location: Pakistan Join Date: Apr 2015 Posts: 11	Suggestion for next version of SeqMonk Hi Simon, Like many others on this forum, I am a big fan of SeqMonk. Here is a suggestion for next version (or may be I dont understand SeqMonk). Yes, we can make customized Annotation Tracks BUT what if I have a specific list of genes involved in a specific process then how do I make annotation track. I guess there is room for improvement here, an option may be included where I can upload a list of genes with a variety of fuctions (names or TAIRids) and then make a customized Annotation Track. If this is added to the SeqMonk next version, that will greatly help my analysis. If there is already one then I am sorry for oversight. I am working with flowering time in Arabidopsis and there are several pathways involved such as Vernalization pathway, Photoperiod Pathway, Ambient Temperature Signalling Pathway, Autonomous Pathway and some Biochemical Pathways, furthermore new genes are discovered on daily basis that add to the flowering time and the TAIR database is not regularly updated. Your kind insight will be highly appreciated. Best Regards Fahim

01-26-2015, 07:43 PM	#283
kentawan Member Location: Singapore Join Date: Apr 2014 Posts: 14	Hi Simon or anyone else, Is it possible to edit the chromosome names in SeqMonk? I started my project using genomes downloaded from Ensembl FTP. As I proceed I realized that I needed data from UCSC Genome Browser (CpG Island estimated position to be exact, which is an annotation track). I realised that the genome labelling is different, hence I want to edit them. Any way of doing it? regards Ziyi

04-23-2015, 07:14 AM	#285
simonandrews Simon Andrews Location: Babraham Inst, Cambridge, UK Join Date: May 2009 Posts: 871	I've just released seqmonk v0.30.0 on the project web site. This version adds an optional link between seqmonk and a local R installation to allow us to easily run some R based analyses seamlessly from within SeqMonk. Initially the applications we've implemented using this link are DESeq2 and EdgeR for RNA-Seq analysis and logistic regression for replicated bisulphite sequencing analysis. We're open to suggestions for what other packages or tests should be implemented so please shout out if there's anything you think would be particularly useful.

04-25-2015, 01:44 PM	#287
simonandrews Simon Andrews Location: Babraham Inst, Cambridge, UK Join Date: May 2009 Posts: 871	The RNA-Seq quantitation has been in for a while, but we now make it easy to link out to DESeq and EdgeR without having to leave the program. For smallRNAs you could still use the RNA-Seq pipeline but just change the feature type you want to quantitate to the appropriate small RNA category (miRNA for example). Given that small RNAs aren't generally spliced though you could just use a normal read count quantitation too. There's also the small RNA QC plot which is a really useful way to start looking at your small RNA data.

04-27-2015, 03:02 AM	#291
simonandrews Simon Andrews Location: Babraham Inst, Cambridge, UK Join Date: May 2009 Posts: 871	Is this RNA-Seq or small RNA? What is the quantitation? Did you do the corresponding QC plot for the data type you're using - if so, what did that look like? You said you did two levels of additional normalisation, were they obviously needed? Did the distribution change much after applying them? You seem to have a very large proportion of unmeasured probes which seems suspicious. Does the raw data look the way you'd expect?

04-27-2015, 03:19 AM	#292
mfahim Member Location: Pakistan Join Date: Apr 2015 Posts: 11	1. is this RNA-Seq or small RNA? Ans.Yes, this is smallRNA seq. 2. What is the quantitation? Ans. As you mentioned previously, I applied the normal RNAseq Quantitation pipeline. 3. Did you do the corresponding QC plot for the data type you're using - if Ans. so, what did that look like? 4. You said you did two levels of additional normalisation, were they obviously needed? Did the distribution change much after applying them? Ans. Yes, as per your youtube instruction I went to check the plot and after application of the two normalization levels. This is how it appears (the shared picture), improved as compared to the previous plot but obviously still bad. 5. You seem to have a very large proportion of unmeasured probes which seems suspicious. Does the raw data look the way you'd expect? The QC plot was having some error, I have sent the report. I have no idea what is happening here.

04-30-2015, 12:33 AM	#295
mfahim Member Location: Pakistan Join Date: Apr 2015 Posts: 11	Bingo!! Thank you Simon for the Genelist.. That is exactly what I was looking for. Perfect. More questions next week... Cheers Fahim

06-22-2015, 06:13 PM	#296
mathew Member Location: australia Join Date: Jan 2011 Posts: 81	Question about Seqmonk annotations I am using Seqmonk 30. I have ran Chip analysis and has annotations like CGI, TSS, mRNA, Promoter etec. I want to export coordinates with all the annotations in one file Is it possible how to get around. Thanks

06-23-2015, 05:51 AM	#298
mathew Member Location: australia Join Date: Jan 2011 Posts: 81	.dat file will give the genome coordinates of that particular gene. If I create a report that will be for one annotation i.e either TSS 2000(say) bp up down / mRNA/ CGI / intergenic and so on. What I want to do is to call peaks and annotate each peak with any region whether it is in enhancer, promoter, CGI, mRNA assigning all criteria in one output file. So I am looking for a file outlined below- chr1 100 1000 geneXX cgi 2000bp enhancer Tss 1000 bp RPKM 1 RPKM2 Diff

07-01-2015, 08:23 AM	#299
simonandrews Simon Andrews Location: Babraham Inst, Cambridge, UK Join Date: May 2009 Posts: 871	We've just released seqmonk v0.31.0 onto the project web site. We've added some new options for analysing RNA-Seq and BS-Seq data and have tidied up the user interface for cases where you have long names for your data stores or probe lists. http://www.bioinformatics.babraham.a...jects/seqmonk/

09-09-2015, 08:21 AM	#300
simonandrews Simon Andrews Location: Babraham Inst, Cambridge, UK Join Date: May 2009 Posts: 871	SeqMonk v0.32.1 has just been released. This fixes a bug which meant that on some systems the dialog which showed the progress of R scripts could hang. We've also taken the opportunity to improve the reporting of R crashes so that the full R log is now also sent alongside the crash report, which should help to sort out any remaining R issues. We also improved the line graph, adding the ability to click on a line to see which probe it came from, and double click on it to go to that probe in the chromosome view.