Kai Lao, Applied Biosystems - “Deep Sequencing-Based Whole Transcriptome Analysis of Single Early Embryos”
I think all sequencing was done with ABI SOLiD.
To get answers about early life stages, you need to do single cells – early life is in single cells, or close to it. When you separate a two cell embryos, miRNAs are symmetrically distributed (measured by array). T1 and T2 have similar profiling. When you separate in 4 cells – it's still the same....
Can you do the same thing with next gen sequencing to do whole transcriptome? (Yes, apparently, but the slide is too dark to see what the method is.) Quantified cDNA libraries on gel, then started looking at results.
If you do everything perfectly, concordance between forward and reverse strand should be same. However, if you do the concordance between two blastomers, you see different results. [not sure what the difference is, but things aren't concordant between two samples....]
First, showed that libraries have very high concorndance – same oocyte gives excellent concordance. However, between dicer knock out and wt, you get several genes that do not have same expression expression in both. Many genes are co-up-regulated or co-down-regulated.
One gene was Dppa5. In wt, it had low expression, in Dicer-KO and ago2-KO, they were upregulated.
After Dicer Genes were KO at day 5, only 2% of maternal miRNAs survived in a mature Dicer KO oocyte (30 days.) Dicer-KO embryos can not form viable organisms (beyond first few cell stages.)
Deeper sequencing is better. With 20M reads, you get array level data. You get excellent data beyond 100M reads.
No one ever proved that multiple isoforms are expressed at the same time in a cell – used this data to map junctions, and showed they do exist. 15% of genes expressed in a single cell as different isoforms.
To get answers about early life stages, you need to do single cells – early life is in single cells, or close to it. When you separate a two cell embryos, miRNAs are symmetrically distributed (measured by array). T1 and T2 have similar profiling. When you separate in 4 cells – it's still the same....
Can you do the same thing with next gen sequencing to do whole transcriptome? (Yes, apparently, but the slide is too dark to see what the method is.) Quantified cDNA libraries on gel, then started looking at results.
If you do everything perfectly, concordance between forward and reverse strand should be same. However, if you do the concordance between two blastomers, you see different results. [not sure what the difference is, but things aren't concordant between two samples....]
First, showed that libraries have very high concorndance – same oocyte gives excellent concordance. However, between dicer knock out and wt, you get several genes that do not have same expression expression in both. Many genes are co-up-regulated or co-down-regulated.
One gene was Dppa5. In wt, it had low expression, in Dicer-KO and ago2-KO, they were upregulated.
After Dicer Genes were KO at day 5, only 2% of maternal miRNAs survived in a mature Dicer KO oocyte (30 days.) Dicer-KO embryos can not form viable organisms (beyond first few cell stages.)
Deeper sequencing is better. With 20M reads, you get array level data. You get excellent data beyond 100M reads.
No one ever proved that multiple isoforms are expressed at the same time in a cell – used this data to map junctions, and showed they do exist. 15% of genes expressed in a single cell as different isoforms.
Labels: AGBT 2009
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