[Skissors]

Filtering by Statistical Test

Hi,

I used the RNA-seq pipeline in SeqMonk to process my data. My initial probe list was 26127 in size. So I performed a filter by statistical test using intensity difference. In the pop-up box where it indicates "Probes per sample", when I try to input 26127, it defaults to 13036. Is there any reason why it would default to this value?

I'm new to using SeqMonk and NGS in general, so I have much to learn.

Thanks for your help!

[simonandrews]

Two related questions:
1.Can we used FAstqc for qc check of bisulfite seq data. Will it give me CG ratios etc?

Yes. FastQC reports provide some useful information in analysing BSSeq. All of the normal quality stuff is the same as any other library, and the sequence composition parts are informative - although since BS-Seq libraries are expected to look odd you will get error flags on the composition based modules (esp per sequence composition) since there should be very few Cs and lots of Ts.

There is also a lot of more BS-Seq specific QC in the bismark reports which you should definitely look at. This will cover things like the BS strands which were found, the overall level of methylation in different contexts and positional biases in methylation (M-bias plots).

2. I have used Seq monk but have not used Bismark. Is there a GUI version Bismark equivalent to Seqmonk?
Thanks

No, the nature of the computation needed for bismark means that it's not really feasible to run this on a normal desktop computer. There is a galaxy wrapper for bismark which can make it easier for people who really want to avoid the command line altogether, but the bismark command options are pretty simple for normal libraries. I suppose we could write a small graphical program to help to set this up, but it would just end up constructing a normal command line at the end.

[mathew]

FastQc for Bisulfite seq

Two related questions:
1.Can we used FAstqc for qc check of bisulfite seq data. Will it give me CG ratios etc?
2. I have used Seq monk but have not used Bismark. Is there a GUI version Bismark equivalent to Seqmonk?
Thanks

[simonandrews]

Hi Simon,

when I have a genome with pseudo chromosomes, the "go to position" feature only shows pseudo chromosomes. Could that be expanded to actually allow for going to scaffold coordinates also? Or is there a work around to go to actual scaffold positions?

Cheers

That's a really good point. I'll see if there's an easy way to add the original scaffolds into the options for that.

[ShellfishGene]

The video shows how to build both conventional genomes, but also how to make pseudo chromosomes when you're working with assemblies which are incomplete and may contain many thousands of scaffolds.

Hi Simon,

when I have a genome with pseudo chromosomes, the "go to position" feature only shows pseudo chromosomes. Could that be expanded to actually allow for going to scaffold coordinates also? Or is there a work around to go to actual scaffold positions?

Cheers

[simonandrews]

Hi Simon,

I ran ChIP seq analysis and created a wiggle file to look for peaks. Can I export the wiggle track file or not at all.
Thanks

Yes, you can export the data in a couple of ways.

If you want to put the data into a genome browser or a general genome visualisation tool like IGV then you can do File > Export Current View > BEDGraph, which will export the data as a BEDGraph file. We have to use BEDGraph format rather than wig since the probes in SeqMonk are not guaranteed to be fixed width.

If you want to just get the data out in a format which is easy for you to manipulate then you can create a report using the options in the report menu. Doing an annotated probe report (even if you don't actually annotate) is probably what you'd want to try first.

Hope this helps.

[mathew]

CAn I save wiggle file

Hi Simon,

I ran ChIP seq analysis and created a wiggle file to look for peaks. Can I export the wiggle track file or not at all.
Thanks

I've uploaded a new release of SeqMonk to the project web site. Version 0.27.0 makes some improvements to the RNA-Seq quantitation as well as adding a new tool which makes it easy to automatically split large numbers of samples into the appropriate data groups or replicate sets.

We've also improved the re-import tool so that you can now down-sample a large dataset to a size of your choosing, or filter the reads by their length.

Finally we've fixed a couple of bugs, notably a problem with multiple testing correction when analysing large numbers of HiC probes, and one which prevented custom genomes created from GFFv3 files from automatically loading the annotation in the new genome.

Please let us know if you have any problems with the new version.

[crazyhottommy]

Thank you for developing this excellent tool!

I've uploaded a new release of SeqMonk to the project web site. Version 0.27.0 makes some improvements to the RNA-Seq quantitation as well as adding a new tool which makes it easy to automatically split large numbers of samples into the appropriate data groups or replicate sets.

We've also improved the re-import tool so that you can now down-sample a large dataset to a size of your choosing, or filter the reads by their length.

Finally we've fixed a couple of bugs, notably a problem with multiple testing correction when analysing large numbers of HiC probes, and one which prevented custom genomes created from GFFv3 files from automatically loading the annotation in the new genome.

Please let us know if you have any problems with the new version.

[simonandrews]

I've uploaded a new release of SeqMonk to the project web site. Version 0.27.0 makes some improvements to the RNA-Seq quantitation as well as adding a new tool which makes it easy to automatically split large numbers of samples into the appropriate data groups or replicate sets.

We've also improved the re-import tool so that you can now down-sample a large dataset to a size of your choosing, or filter the reads by their length.

Finally we've fixed a couple of bugs, notably a problem with multiple testing correction when analysing large numbers of HiC probes, and one which prevented custom genomes created from GFFv3 files from automatically loading the annotation in the new genome.

Please let us know if you have any problems with the new version.

[simonandrews]

I've uploaded a tutorial video showing how to use the custom genome builder tool in SeqMonk to easily work with genomes which aren't available in our core database. The video shows how to build both conventional genomes, but also how to make pseudo chromosomes when you're working with assemblies which are incomplete and may contain many thousands of scaffolds.

[simonandrews]

I'm going to make up a tutorial video to show the process of making one of these custom genomes to try to explain this a bit better. It's probably not obvious from just the documentation and it's a very new feature so a few people will be trying this out.

[tirohia]

That worked perfectly and I doubt I would have thought of that straight off the bat. I'm am indebted to you

many thanks.

[simonandrews]

So, yes, that appears to work. Now it's a whole bunch of errors about certain reads mapping somewhere past the end of the chromosome (Reading position 14320062 was 2642bp beyond the end of chr25 (14317420)).

Not sure how that's possible given that it's all coming from the same set of data, off to check it all again though.

Cheers
Ben.

If you've just built the genome from the GTF file them the chromosome will end where the last feature finishes, so you will get reads mapping off the end of the chromosome. If you want to get the chromosome lengths correct then you can either fix them manually when you build the genome or you can pass in the fasta files in addition to the GTF file to get the full chromosome length.

[tirohia]

So, yes, that appears to work. Now it's a whole bunch of errors about certain reads mapping somewhere past the end of the chromosome (Reading position 14320062 was 2642bp beyond the end of chr25 (14317420)).

Not sure how that's possible given that it's all coming from the same set of data, off to check it all again though.

Cheers
Ben.

[simonandrews]

Hi Ben,

You should be fine if you map against the chromosome sequences. You'll need to use a splice aware mapper such as tophat to do the mapping, but you can also pass in your GTF file to the mapper so that it will effectively map against the transcriptome first, but will give you genomic coordinates.

Let me know if it works out OK, but hopefully this batch of mapped reads will be OK.

Simon.