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Hi Dear, I want to run a de novo transcriptome project by Roche 454 with a species of fish cDNA library that its genome size is about 3*10^9, which kind of picotiter plate region is better for me and why? 1/2 or 1/4 or 1/8 ?
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If the genome is 3E9 bp (similar to Homo sapiens)), then the transcriptome will be at least 3E6 bp. I'm not sure even 1/2 plate will have enough yield to cover your transcriptome, as there will be > 1000x difference between some transcript yields. You may need to use Illumina or Proton instead. -
I have run a devo transcriptome assembly using 454 on a fish with a similar sized genome (slightly larger than human genome). I ran 4 individuals in a whole 454 FLX run.
i used 3' targeted cDNA (however I seem to remember reading that for some reason this isnt the ideal protocol when using 454...?).
If memory serves I got around 30,000 contigs when aligning to a closely related reference transcriptome, and a bunch more via de novo assembly. From the 30, 000 contigs there were ~50K SNPs/indels identified....around 9K of these suited my criteria for a high quality putative SNP.
In theory 3' target cDNAs should increase yield of homologous fragments between individuals, and a full 454 plate seemed to give us good representation of the transcriptome.Last edited by JackieBadger; 10-07-2012, 11:43 AM.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.Yesterday, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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