Dear all,
some time ago we did Taqman for a couple of candidate genes on some human samples, that showed downregulation of these genes. We therefore decided to go for a low-depth RNA-seq on these 38 samples, but it turns out that the results are no near what we see with Taqman where we used verified probes from Life Technologies.
We are a bit lost now and dont know what to trust. I would appreciate any input! Thanks a lot.
Info about sequencing process:
19-plexing 2x50bp strand-specific on a HiSeq2500.
FastQC files showed great quality but a bit of contamination of index sequences, so I trimmed the reads with TrimGalore. Ran FastQC again and everything was great. I mapped to hg19 (UCSC from iGenome) using the Tophat 2.0.8. I get around 7-10 million mapped reads.
I then continued with Cufflinks, Cuffquant and Cuffnorm (without de novo discovery). I also tried using DESeq 2. Get about the same results (also independent on whether I use classic.fpkm, quartile og geometric normalization).
Thanks a lot - appreciate your input!
some time ago we did Taqman for a couple of candidate genes on some human samples, that showed downregulation of these genes. We therefore decided to go for a low-depth RNA-seq on these 38 samples, but it turns out that the results are no near what we see with Taqman where we used verified probes from Life Technologies.
We are a bit lost now and dont know what to trust. I would appreciate any input! Thanks a lot.
Info about sequencing process:
19-plexing 2x50bp strand-specific on a HiSeq2500.
FastQC files showed great quality but a bit of contamination of index sequences, so I trimmed the reads with TrimGalore. Ran FastQC again and everything was great. I mapped to hg19 (UCSC from iGenome) using the Tophat 2.0.8. I get around 7-10 million mapped reads.
I then continued with Cufflinks, Cuffquant and Cuffnorm (without de novo discovery). I also tried using DESeq 2. Get about the same results (also independent on whether I use classic.fpkm, quartile og geometric normalization).
Thanks a lot - appreciate your input!
Comment