Gene Expression

[elg]

Hi there,

It seems most of the people here are using the Solexa platform for genomic sequencing. But what about gene expression?
Might be useful to share with others what strategies/tools are you using to annotate and analyze all the SAGE tags coming out of the sequencer. Illumina provides annotation files for human and mouse, but how are people dealing with other species with and without an available genome?

Cheers,
elg

[ScottC]

Yeah, that's an interesting area... I'd like to hear about that too.

I'm looking forward to a Nature Methods paper that should come out from the
Sanger shortly about bacterial expression studies using concatenated bacterial cDNA. There's not many people doing bacterial transcriptomics yet! Anyone here doing it?

[seidel]

We've been using the platform for gene expression. I can't say it's all that useful yet, because basically the counts for genes fall across 4 orders of magnitude, with many counts in the 10s to 100s. It's hard to compare 10 counts of YFG (your favorite gene) in one sample to 50 in another, and really believe that's a 5-fold difference, especially when the scatter in the 0-100 count range is so high among replicates.

Illumina's tables for human and mouse are out of date and they have no plans to replace them or regenerate them. But there are methods for generating the tables yourself. There's a thread and some resources over at google groups: http://groups.google.com/group/solexa

-Chris

[elg]

Thanks for the link, Chris, that was helpful!

about the scatter in the 0-100 count range, I have observed the same behaviour in my data. But shouldn't this be a Poisson distribution matter and then just check whether the count differences are significant or not?

Esther

[ScottC]

This might be interesting to you guys:

"Ultra high-throughput sequencing is emerging as an attractive
alternative to microarrays for genotyping, analysis of methylation
patterns and identification of transcription factor binding sites. Here,
we describe an application of the Illumina sequencing platform to study
mRNA expression levels. Our goals were to estimate technical variance
associated with Illumina sequencing in this context and to compare its
ability to identify differentially expressed genes with existing array
technologies. To do so, we estimated gene expression differences between
liver and kidney RNA samples using multiple sequencing replicates, and
compared the sequencing data to results obtained from Affymetrix arrays
using the same RNA samples. We find that the Illumina sequencing data
are highly replicable, with relatively little technical variation, and
so, for many purposes, it may suffice to sequence each mRNA sample only
once (i.e., using one lane). The information in a single lane of
Illumina sequencing data appears comparable to that in a single array in
enabling identification of differentially expressed genes, while
allowing for additional analyses such as detection of low-expressed
genes, alternative splice variants, and novel transcripts. Based on our
observations, we propose an empirical protocol and a statistical
framework for the analysis of gene expression using ultra
high-throughput sequencing technology"

http://www.genome.org/cgi/content/abstract/gr.079558.108v2

[lichun]

sample amount needed

That's really good news.
Would anyone give me some suggestion about the minimal RNA sample needed for reliable and repeatable solexa sequencing ? Since I attemp to
deal with the transcriptome of very tiny sample.

Thanks a lot!
Lichun

This might be interesting to you guys:

"Ultra high-throughput sequencing is emerging as an attractive
alternative to microarrays for genotyping, analysis of methylation
patterns and identification of transcription factor binding sites. Here,
we describe an application of the Illumina sequencing platform to study
mRNA expression levels. Our goals were to estimate technical variance
associated with Illumina sequencing in this context and to compare its
ability to identify differentially expressed genes with existing array
technologies. To do so, we estimated gene expression differences between
liver and kidney RNA samples using multiple sequencing replicates, and
compared the sequencing data to results obtained from Affymetrix arrays
using the same RNA samples. We find that the Illumina sequencing data
are highly replicable, with relatively little technical variation, and
so, for many purposes, it may suffice to sequence each mRNA sample only
once (i.e., using one lane). The information in a single lane of
Illumina sequencing data appears comparable to that in a single array in
enabling identification of differentially expressed genes, while
allowing for additional analyses such as detection of low-expressed
genes, alternative splice variants, and novel transcripts. Based on our
observations, we propose an empirical protocol and a statistical
framework for the analysis of gene expression using ultra
high-throughput sequencing technology"

http://www.genome.org/cgi/content/ab...r.079558.108v2

[bioinfosm]

Hi Elg and Seidel,

Would you be able to share some information about running the GA pipeline on Solexa 3' tag data? Eland can be used on latest+squashed tag tables for the 17 cycles? Or the 4bp tag should be included as well?

Any pipeline or scripts that can be made use of for the analysis. ELAND_TAG is one way I looked up, that even gives the counts. Have you tried it out, and any help in setting that up?

Thanks..

[elg]

Hi bioinfosm,

no, you don't have to add the 4bp tag to the tag tables. I used Eland in tag modus as it is described in the user's manual, no special changes. But since Illumina only offers tag tables for human and mouse we are currently setting up our own pipeline.

Esther

[bioinfosm]

You might want to look at this for a pipeline to make tag tables

http://research.stowers-institute.org/microarray/tag_tables/index.html

I am not sure why we have 18 cycles for the DpnII, as I thought its 16/17. SO do I simply use that many cycles in the config for GERALD?