I just got data back from the sequencing center and I am unsure if I should be concerned that duplicates of the same sample have very different read counts. To explain, I asked the sequencing center to include duplicates of 3 of my samples (of 250 total) in the libraries to use as quality control. My average read count is 470k with a standard deviation of about 50k. However, the duplicated samples differ by 10k to 270k from themselves. Should I be concerned at this variation? And what might be driving the difference?
A. Z. Andis Arietta
The duplication in your case is the replication of the sample this means technically replication are independent samples in terms of sequencing with different barcodes and pooled together among other samples. So there are many factors which can affect the number of reads in final output data. I don't think concentration is the primary factor since the libraries are generally pooled in normalized concentrations and I don't think a sequencing facility/company would do mistake in this step. The other reasons which are technical reasons a not in our control are the cluster generation, sequencing, cluster determination and demultiplexing etc.
Think it in this way, all the samples were sequenced on the same platform, with the same number of sequencing cycles, still, the number of reads in the final output is not the same.
And if you should be concerned? This is you to decide if you do not see what you expect in those duplicate samples and if they show different trends. I would recommend you to see the trend and relative abundances of the final processed data instead of thining about the size of the raw data.
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