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Old 02-25-2011, 07:49 AM   #1
Dan_cham
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Default suggestion and price

hello

I intend to sequence the entire Exome of a certain vertebrate with X10 coverage.

assuming that his Exome is the same size as human Exome (about 75,000,000 bp), can someone give me an estimation of price and maybe recommendation for places that do Solid?

Thanks so much

Dan
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Old 03-03-2011, 10:08 AM   #2
boscoliveira
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Many companies do whole exome sequencing for humans, please check online. Edge Bio is one with 5 or 6 Solid machines (I don't work there, am not endorsing). However, as far as I'm aware, commercial baits are sold only for human and mouse exomes. You'd have to design your own set and have it synthesized, if the genome of the animal is known.
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Old 03-03-2011, 10:38 AM   #3
Dan_cham
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thanks for the reply

what do you mean when u say "baits"?

my animal is one with unknown genome. i intend to do de novo assembly.

thanks again

Dan
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Old 03-03-2011, 10:42 AM   #4
boscoliveira
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To capture the exonic regions of a genome (exome) one has to design and print RNA baits that will anneal and "capture" the desired exons. This cannot be done without knowledge of the genomic sequence. In this situation you have to sequence the whole genome, preferably using a platform with long reads, such as the 454, but that can be very costly.
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Old 03-03-2011, 10:48 AM   #5
Dan_cham
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To my understanding, i am going to send RNA samples - they will transfer it to cDNA, and will enrich for poly- A, then they will attached adapters which will allow them to sequence the Exome with out knowing the entire genome
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Old 03-03-2011, 10:54 AM   #6
boscoliveira
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Oh, you're talking about the transcriptome, not the exome. It is quite a different matter, and I am not aware of this being useful for a de novo sequencing project. It is impossible to reconstruct a genome from a transcriptome, as far as I know.
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Old 03-03-2011, 11:02 AM   #7
Dan_cham
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Sorry i didn't know there's a different.

my only intent is to assembly the transcriptom in order extract (after assembly) many genes related to my research.

I know about "trans abyss" that do de novo assembly for transcriptom

Do it make more sense now?
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Old 03-03-2011, 10:21 PM   #8
Pepe
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10x is very little sequencing for a de novo project.
Also, consider normalization of your samples to obtain even coverage the transcripts.
Prices depend on the facility where you sequence. Having long reads and paired end reads will help de novo assembly, so 454 or Illumina seem better choices than Solid.
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Old 03-04-2011, 03:09 AM   #9
colindaven
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I'd go for Illumina as well with long PE reads as well. 454 would be a lot more costly for a vertebrate.

10x average coverage is impossible to predict and get for a transcriptome since you don't know which genes are being transcribed, there have been other discussions on this forum about this topic recently.

Good luck, and have a look at Velvet too.
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Old 03-04-2011, 03:42 AM   #10
Dan_cham
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I will do PE reads, with rRNA reduction kit and POLY-A enrichment kit.

The center committed for 40 million reads 85 bp each, now assuming that the transcriptom is then 75 million bp (has in human) or less, I will get (theoretically) very good coverage.

by the way the price they offer to me is 3500$, good price?

Thanks for the replies...
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Old 03-04-2011, 03:48 AM   #11
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Just to be clear, in any given tissue, under any give conditions, only a fraction of the total genes of an organism are going to be expressed. Further some genes will be expressed at much higher levels than others.

So this "10x" coverage of a transcriptome terminology is highly misleading because it conflates two different sample types: genomic DNA (same in every cell of an organism) and RNA (different).

--
Phillip
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Old 03-04-2011, 05:07 AM   #12
Dan_cham
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you are correct, and i am aware of this.

my intention is to get a minimum coverage of X10 after the analysis and assembly. so of course i will need much higher coverage that will take into account differentiation expression (certain genes that express higher then other). luckely the genes I want to find are suppose to express in high level consistently. other genes that I will get are like a bonus to me for future research about the organism.

Thanks
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