nodalpoint.org - Genomics - Comments

Solexa

chris — Mon, 21 May 2007 15:38:45 -0400

Solexa's technology produces even shorter reads - approx 30bp or so. It seems to be rapidly gaining in currency and analysis effort, if the Cold Spring Harbor meeting I attended earlier this month is any indication. A number of the large sequencing centers are choosing which way to split, and rather predictably both Solexa and 454 are getting attention. It will be interesting to see whether one emerges as a winner, or as suggested below, the combination will prove more powerful.

It is certainly becoming clear that these technologies are only likely to be useful in resequencing and EST-like projects where reference genome scaffolds already exist for assembly. I'm sure someone will come up with a clever work-around eventually, though.

More like these to come

badboyz — Fri, 11 May 2007 04:40:11 -0400

I think more and more people will be getting into this strategy at the beginning because 454's method much to be desired. On the other hand I think we should look carefully at how ABI is going to handle this new and emerging market. Their new high-throughput sequencer, SOLID, is based on the Agencourt sytem.

What would be really cool is if we could get a machine that could interchange modes. Quite an instrumentation challenge because one technology is bead-based (454) - not sure about SOLID - and the other is clone based (Sanger).

Bead Events ???

badboyz — Fri, 11 May 2007 04:28:36 -0400

I work with this data every day and let me say that it's a bit of pain and pleasure. The data management issue is something most of us dont have a problem with, but the pain point comes with having to continually refine our analysis to handle data inconsistencies.

On the whole 454 are making a great effort to deliver clean datasets to their customers. The longer reads do help although I'm a bit wary of these homopolymers and phase errors. And then there are these "Bead Events" that I keep on hearing about from various people. Personally, I dont think that we should ever expect too much from a new technology that is trying to take massive strides too soon. I like the idea of combining data sources together e.g Sanger + 454, Solexa + Sanger + 454, etc...

Mostapha Ronaghi, the inventor of the first pyrosequencing system, gave quite an informative talk in Malaysia about technologies, the state of the art, cost issues, etc. It can be streamed online from
http://www.mgrc.com.my/eLectureRonaghi.shtml.

Back to the data issue. Does anybody have more insights in what kind of inconsistencies or other things that people may need to be wary of?

Upgrade to 250bp in the works?

lbbros — Thu, 01 Feb 2007 02:42:28 -0500

As far as I know, the 454 people promised an "upgrade" of their technology to extend the maximum reads to 250 bp, I believe. Of course, I want to see it before I believe it.
However, I will be able to confirm in a few months, as the people responsible for the 454 instrument here want to perform that upgrade.

shorts reads

maximilianh — Wed, 31 Jan 2007 05:35:43 -0500

short reads are less a problem if one has a close genome at hand that has already been sequenced... the more genomes are available the easier it is to simply assemble by alignment, which is similar to what ensembl is doing now with their 3x genomes (see their 2007 article in NAR).

but anyways, yes, I was rather thinking about re-sequencing than sequencing from scratch.

Short reads

Neil — Tue, 30 Jan 2007 20:19:32 -0500

I just came out of a group meeting where 454 was discussed. Something I hadn't realised is that the read length is very short - 100 bp or so. This makes 454 great for resequencing, mapping reads onto existing assemblies and finishing, not so great for assembly of a genome from scratch. I'm excited by the idea that any lab could do a genome one day, but it won't be for a while yet.

I vaguely recall a technology where single DNA strands are fed through nanopores and changes in electrical resistance used to read bases? If anyone has a better memory than mine, feel free to comment. That kind of technology strikes me as "the way forward" for truly high-throughput sequencing.

We have one of those

lbbros — Tue, 30 Jan 2007 18:16:15 -0500

I can say that handling the data from that beast is quite a task, according to my co-workers that rebuild and analyze the instrument's reads to get clean and (possibly gap-free) sequences.

In my opinion pyrosequencing such as what the 454 system does will not be a viable option for many institutes until there is this large problem to tackle. At first we had just one person working on the data, but it became evident that it wasn't enough. Now we have three people dedicated to the data analysis, on different projects.

Cool does it for me

Neil — Fri, 08 Apr 2005 03:44:30 -0400

It's true that many people will overlook this, because (a) it doesn't involve medicine or humans and (b) it's in a fledgling and open access journal. Which is a shame. "Wow, that's cool" should be enough for any real scientist, IMHO.

I'm a big fan of the BMC journals (Bioinformatics, Genomics and Genome Biology).

re: dna preps

Neil — Fri, 08 Apr 2005 03:41:18 -0400

Presumably you get a bunch of fruit flies, gently mash them and extract DNA from the mess. If anything else is living inside the flies, you will get DNA from that too. How much of that DNA you end up cloning and sequencing then depends on the factors that you highlighted in the comment. Plasmid/E. coli DNA is screened out during the genome assembly (e.g. using cross_match).

I think this aspect of a sample containing DNA from other sources is one that people tend not to think about - which is one of the reasons that I found the paper of interest.

From the paper:"The amount of

Greg — Fri, 08 Apr 2005 03:26:07 -0400

From the paper:

"The amount of endosymbiont DNA present in a genome deposited in the Trace Archive depends on several factors: the number of sequences generated by the project, the size of the host genome, the size of the endosymbiont genome, and the number of copies of the endosymbiont present in each cell of the host."

I would now like to demonstrate my ignorance of DNA preparation techniques. Endosymbiont here refers to integrated Wolbachia DNA in the fruit fly DNA or fruit fly cell ? Does this mean the trace archive is picking up non-intracellular parasite/organismal DNA, or is that somehow excluded during the DNA extraction process ? Also how effective are the screens for plasmid/E. coli DNA ?

Marketing

Greg — Fri, 08 Apr 2005 03:18:21 -0400

It is quite impressive that mountains of data, full of potentially interesting discoveries, are just lying about in some NCBI server room. While the Wolbachia genomes study certainly demonstrates this, it just isn't marketable enough to spread the message. I'm only half joking here, I know that to the people that understand this stuff marketing is the last thing they would be thinking about. However it seems a shame that the "wow! that's kinda cool" aspect of this discovery (including the methods) will not be widely absorbed. I look forward to the next paper of the month, takers ?

Application for Milano

ranrub — Tue, 16 Nov 2004 05:50:21 -0500

Let me give you an example. When performing a microarray experiment in which you test expression response to p53 (a DNA damage-related transcription factor and Very Important Protein), you get a list of 150 genes that were significantly up- or down-regulated. Now you want to find information about these genes. Milano enables you to perform automatic searches in the literature for these genes and cross them with experiment-specific search terms.
For example, searching these genes with the term 'p53', will give you positive hits on genes that co-appeared in articles with their putative regulator (as detected in the microarray experiment). This enables you to find known p53 targets, or even regulatory feed-back loops - if you find regulators of p53. Other intersting applications can be thought of.

Further development

ranrub — Tue, 16 Nov 2004 05:20:36 -0500

Currently Milano is in 'alpha'. I plan to release the source (with a proper open-source license) once I clean it up .

Further advancements will include accepting different types of accession numbers, such as Affymetrix probeset IDs. Currently I use Locuslink/Gene Id's because I feel they have the most meaning for this type of search, and because GeneRIF is based on these genes. Also, most microarray vendors supply LocusLink IDs in their annotations.

Feature suggestions will be gladly accepted!

duplicate

Neil — Tue, 16 Nov 2004 00:38:17 -0500

This story appeared recently then disappeared - I promoted it back to the front page. Don't know if that caused any problems.

I have little/no microarray knowledge, but a problem I have with this kind of approach is that it's not clear what the service is meant to do or what I should be typing in as search terms. Perhaps I would have a better idea if I did microarray experiments. I assume the idea is to correlate gene annotation data with expression data? In which case I can think of better ways than small, limited searches - but then I am a proponent of "all against all" approaches.

Admin

Greg — Mon, 15 Nov 2004 06:31:03 -0500

I haven't done a word for word comparison, but I assume this story is a duplicate of the story in the queue ? I will delete the one in the queue if that is okay, if not let me know.

Milano looks interesting, but unfortunately I don't have much expertise in this area to comment seriously. Do you plan on developing it further ?