[radekchr]

Bumping to ask a seemingly simplistic question:
I am a biologist with the desire to learn all there is to know about NGS analysis, but my problem has been finding a good source of information that starts at the VERY beginning. If the criticism is biologist should learn to think more like bioinformaticians, then why has it been so difficult for me to do just that? Am I just looking in the wrong places? Is there a web course out there that will teach me the ins and outs of the different alignment programs and analysis softwares using the cloud or otherwise?

Hey 1230Rock,

Here at MSU, there is a 2 week summer program for biologists that goes over the basics of a lot of handling of next-gen sequencing data and using Amazon cloud computing.
Here's a link to last year's course, which also provides a link to the exercises we did last year. Not sure this starts at the VERY beginning, but I thought it was useful nonetheless!

http://bioinformatics.msu.edu/ngs-summer-course-2011

[1230Rock]

Bumping to ask a seemingly simplistic question:
I am a biologist with the desire to learn all there is to know about NGS analysis, but my problem has been finding a good source of information that starts at the VERY beginning. If the criticism is biologist should learn to think more like bioinformaticians, then why has it been so difficult for me to do just that? Am I just looking in the wrong places? Is there a web course out there that will teach me the ins and outs of the different alignment programs and analysis softwares using the cloud or otherwise?

[ETHANol]

Novice, I think you think the problem is a little more simple then you think. Imagine giving a 'bioinformatician' a set of pipetmen and asking him to plan an RNA-seq experiment, generate the material, collect the samples and prepare the RNA/libraries. You give him some ready made kits with manuals and tell him to go at it.

Take some time to learn what you need to to process the data correctly. We are not talking about a lot of time, but you will be better off and more efficient if you do. Biology is getting more and more computational, learn and evolve with it, it will make you a better scientist.

Cloud computing, I don't get it. A new PC with a lot of RAM and two big hard drives is so much easier.

[simonandrews]

True that may be, but as NGSfan pointed out, the analysis scene is constantly changing. Say that it takes 6-12 months to make a tool user friendly. A new and better tool will come out about as often, so the user-friendly GUIs will always be one generation behind.

Conversely the problem with always using the new and shiny command line tools is that people too often blindly assume that the results they produce must be reliable because they're using the latest and greatest thing.

The benefits of having a nice GUI are that it allows you to visually explore your data so you can put the analysis results you have into some kind of context. You can do this with command line tools, but I guarantee that most people who run them never bother. Personally speaking I'd rather have a simpler analysis which gives me results I can understand and trust over a more complex analysis which gives me some more hits, but where I'm just trusting that the author's assumptions about the data are valid in my case. There are times when getting every last hit you can out of your data is of primary importance, but a lot of the time people only follow up the really strong hits anyway, and being able to understand where those hits came from and trust them is a big advantage.

My time is pretty evenly split between working on a command line and in a GUI, but I'm very confident in saying that my understanding of our data has been advanced far more by exploring it in a GUI than by looking at the
output of command line tools.

[dahlo]

What still amazes me is that people who are prepared to spend tens of thousands of pounds on sequencing won't stump up a thousand pounds for a beefy PC to help with the analysis. It's painful to see a postdoc wasting their time watching analyses run slowly on a 4 year old PC, which could be done in a fraction of the time if they spent a modest amount on a new machine.

I am also amazed by this fact. Even whole genome sequencing of 2-3 samples is cheap compared to having 1 person employed for a year. Still some people reason that time is something that can be wasted without thinking twice..

[simonandrews]

Also I disagree with the above post. Cloud computing with easy to use GUI graphics will likely take over all NGS analysis very soon. Illumina is already working on this and I'm sure dozens of other companies are too.

I'm sure they are, but I can't see that ending up being the solution. People seem to assume that any NGS analysis requires huge amounts of compute power, which just isn't true for many (even most) applications. A decent high-end PC with a good chunk of RAM is enough for mapping and analysing most types of experiment. I'm sitting in front of my fairly standard Dell Optiplex windows pc which currently has a GUI open showing me half a billion reads, along with derived quantitations and statistical analysis results and not a bit of cloud in sight. Just because it's possible to write an inefficient Perl script which will

Sure, there are types of analysis for which serious hardware is required (assembly of large genomes would be the obvious example), but you can't just go on to amazon and fire up a cloud instance of a machine with 500GB+ of RAM. There are also instances where you would benefit from more processing power (mapping reads to existing genomes), where the cloud can provide this power, but then you end up being limited by the rate at which you can upload data and download results (we're still on a 20Mb connection, but even 100Mb which is pretty standard is very slow for this).

Where the cloud could be very useful is in the reanalysis of data which is already uploaded, to save you the bother and time of pulling this down to your local machine, but there are still great advantages to keeping your local data local.

What still amazes me is that people who are prepared to spend tens of thousands of pounds on sequencing won't stump up a thousand pounds for a beefy PC to help with the analysis. It's painful to see a postdoc wasting their time watching analyses run slowly on a 4 year old PC, which could be done in a fraction of the time if they spent a modest amount on a new machine.

[dahlo]

Also I disagree with the above post. Cloud computing with easy to use GUI graphics will likely take over all NGS analysis very soon. Illumina is already working on this and I'm sure dozens of other companies are too.

True that may be, but as NGSfan pointed out, the analysis scene is constantly changing. Say that it takes 6-12 months to make a tool user friendly. A new and better tool will come out about as often, so the user-friendly GUIs will always be one generation behind.

[NextGenSeq]

You must not have demoed (sp?) CLC Bio very long. All the cons you listed it can do.

Also I disagree with the above post. Cloud computing with easy to use GUI graphics will likely take over all NGS analysis very soon. Illumina is already working on this and I'm sure dozens of other companies are too.

[NGSfan]

Hi,

I'm a bench biologist with a load of RNA-seq data, who is trying to find a good integrated tool for the analysis. I'm neither a bioinformatician nor a mathematician or a statistics fan, so I’m looking for a program where I can put in my data and without getting into much details and complex algorithms, get some answers. With ‘I’, I am sure I represent many other colleagues.

I came from biology and transitioned into bioinformatics. I understand your frustration. But I think it is very naive to think you can get away with doing cutting-edge work with cutting-edge technology and expect everything to be handed to you on a platter.

Perhaps in 5 years more there will be software out there for NGS data to give you answers without you having to *think* about the details and understand how the results are generated.

Becareful of the "cargo cult" mentality, whereby just having a NGS machine will generate you papers. Unfortunately, the HiSeq 2000 does not have a big red button that says "push here for published paper" after dumping your sample into the flow cell.

For groups that have no bioinformatician and no collaborations with such people (sometimes collaborations can take a long long time), it is good that there are companies or goups with an effort to make bioinformatics tools available.

Yes, it is good. But you must be careful not to buy in too readily to the promises of the companies to give you a software package that can do it all. Yes it would it be great not to deal with bioinformaticians and their intellectual rigidness about how to do things properly (and share authorship with them, after all YOU generated all the data).

The sad thing is that if you do not have the ability to analyze your own data then perhaps you shouldn't have been given the funding to carry it out to begin with!

In my opinion, the makers have more or less thrown the user-friendliness out of the window. These softwares are supposed to be made for people with no prior knowledge of bioinformatics, or at least so does the companies behind it claim, but nope. They talk about the algorithms and explain them in a way so that a biologist like me gets lost half the way (this is especially true about the Avadis). And when you finally have passed the aligning step, it needs a whole education to understand and interpret your results.

Well unfortunately Steve Jobs passed away, and I don't see Apple taking over genomic analysis any time soon. Not only that but the methods of analyzing NGS data are constantly changing, improving, and therefore have not had time to settle yet. To expect the companies and the bioinformaticians writing the software for you to not worry about the details (because in reality, the details do matter when methods are not stable) just comes across as very entitled.

Which is another point - in this day and age we should not allow any biologists to finish a degree without some computer science and statistics training. Biology will eventually turn into an information science and if you want to do research you will have to use computers.

Harsh words but this is coming from someone like you who started as a biologist and fought his way into bioinformatics. It's not easy, but for your own career I would strongly urge you to learn - otherwise you will be forever dependent on others to do your analysis - and that means you will be waiting. What will happen when the next technology arrives (and it will), will you be expecting all the software again to be one-click ready for you?

[Strand SI]

Hi,
Do you know if Avadis NGS support Pair end reads generated from Illumina HiSeq? Also do I need to worry about the adapter sequence in my file? Thank you!

Yes, you can import paired end reads generated from any next-gen sequencing platform, including Illumina HiSeq.

Avadis NGS 1.3 will support alignment for raw DNA and small RNA reads. When setting the parameters for alignment, you can choose to trim adaptor sequences.

The Avadis NGS Team
http://avadis-ngs.com

[mediator]

Thank you for trying Avadis NGS (version 1.1.1) and letting us and others know about your experience. It is nice to get such comprehensive feedback on our product – because the more feedback we get, the better and more useful our product can be.

Allow us to comment on some of the issues you described:

First, Avadis NGS doesn’t include an alignment step; it works with pre-aligned data only. With algorithm development for alignment being such a rapidly progressing field, any particular alignment technology incorporated into our software may become outdated quickly. There are many great, free alignment tools available (as some others already pointed out), so we decided to start our software offering from that step onwards.

Second, your discontent with our manual is noted. We are working on making the manual significantly more reader-friendly, in addition to creating step-by-step tutorials for each workflow, so you can more effectively learn about the analyses that are relevant to you without going deep into the algorithms. (You’re quite right about people not reading the instructions for setting up a television, but I’m sure you agree that NGS data analysis is more diverse and complex than that. )

Third, to address the issue you raise about the different expression of the isoforms, see the workflow step “Differential splicing” in the section "Expression Analysis”. This feature will analyze all the different isoforms for a gene and report if one or more of the isoforms shows a different expression behavior. The resulting Entity List with the splicing results can then be visualized with the Gene View (right-click on the entity list and choose “Show in Gene View”) to investigate the results and see the corroborating evidence for the alternative splicing.

Fourth, to investigate trends in the expression values for genes, you can try the “Filter on Parameters” workflows step. This will allow you to find those genes that follow a particular value of a parameter (for instance “Dose” or “Time”). You can set up a numeric experimental parameter and the “Filter on Parameters” workflow step will then show you those genes that show a similar behavior to the trend of you data. (We think this is what you meant with handling trends over the samples).

Fifth, to find the differential expression behavior for a set of genes, you can either load a set of genes from a file or use the “Filter on Entity List” on any results Entity List to create a subset of genes you are interested in. Once a set of target genes is created, any visualization, such as the profile plot, the gene view, or spreadsheet view will then be limited to only the set of target genes you are interested in.

Sixth, we’re unsure what you meant when you wrote that there is no visualization of quality scores, as there are a number of representations for tile/flow/read quality in Avadis NGS’ Quality Inspection features.

Finally, our customer support team is readily accessible by phone and email. They are always happy to help you troubleshoot any issues, even if you are using a trial license at the time. Do not hesitate to contact us directly if you need a better explanation of a particular feature.

Best wishes,

The Avadis NGS Team

Hi,
Do you know if Avadis NGS support Pair end reads generated from Illumina HiSeq? Also do I need to worry about the adapter sequence in my file? Thank you!

[honey]

Avadis

I recently attended a webinar from Avadis and found that it is an excellent tool for downstream analysis for RNA-seq data analysis it has incorporated DEseq for differential expression. I dont work for Avadis but want to share the information

[[email protected]]

Care to join the free data analysis workshop in London next week?

I am on my way to trial the Partek program, so perhaps I could continue on this thread…

Hello,

Thanks for trying out Partek. We've had a load of updates, particularly related to your data analysis queries.
I see you're in London, you are most welcome to sign up for our free workshop and even bring your data along for a deep dat analysis experience.

Hope to see you there!

[steven]

I am on my way to trial the Partek program, so perhaps I could continue on this thread…

Please go ahead, unless you work for them
Thanks for your post, and let us know if you manage to install Galaxy.

[Strand SI]

Thank you for trying Avadis NGS (version 1.1.1) and letting us and others know about your experience. It is nice to get such comprehensive feedback on our product – because the more feedback we get, the better and more useful our product can be.

Allow us to comment on some of the issues you described:

First, Avadis NGS doesn’t include an alignment step; it works with pre-aligned data only. With algorithm development for alignment being such a rapidly progressing field, any particular alignment technology incorporated into our software may become outdated quickly. There are many great, free alignment tools available (as some others already pointed out), so we decided to start our software offering from that step onwards.

Second, your discontent with our manual is noted. We are working on making the manual significantly more reader-friendly, in addition to creating step-by-step tutorials for each workflow, so you can more effectively learn about the analyses that are relevant to you without going deep into the algorithms. (You’re quite right about people not reading the instructions for setting up a television, but I’m sure you agree that NGS data analysis is more diverse and complex than that. )

Third, to address the issue you raise about the different expression of the isoforms, see the workflow step “Differential splicing” in the section "Expression Analysis”. This feature will analyze all the different isoforms for a gene and report if one or more of the isoforms shows a different expression behavior. The resulting Entity List with the splicing results can then be visualized with the Gene View (right-click on the entity list and choose “Show in Gene View”) to investigate the results and see the corroborating evidence for the alternative splicing.

Fourth, to investigate trends in the expression values for genes, you can try the “Filter on Parameters” workflows step. This will allow you to find those genes that follow a particular value of a parameter (for instance “Dose” or “Time”). You can set up a numeric experimental parameter and the “Filter on Parameters” workflow step will then show you those genes that show a similar behavior to the trend of you data. (We think this is what you meant with handling trends over the samples).

Fifth, to find the differential expression behavior for a set of genes, you can either load a set of genes from a file or use the “Filter on Entity List” on any results Entity List to create a subset of genes you are interested in. Once a set of target genes is created, any visualization, such as the profile plot, the gene view, or spreadsheet view will then be limited to only the set of target genes you are interested in.

Sixth, we’re unsure what you meant when you wrote that there is no visualization of quality scores, as there are a number of representations for tile/flow/read quality in Avadis NGS’ Quality Inspection features.

Finally, our customer support team is readily accessible by phone and email. They are always happy to help you troubleshoot any issues, even if you are using a trial license at the time. Do not hesitate to contact us directly if you need a better explanation of a particular feature.

Best wishes,

The Avadis NGS Team