By the way: The effect of seeing a large count for a gene in one sample and zero counts in all other samples is quite common when working with sample derived from very few cells.
In such cases, it is not too uncommon that one only gets maybe around a million mRNA molecules per sample, and after reverse transcription, this is down to possibly less than a hundred thousand cDNA molecules, which then go through many rounds of PCR. If one then sequences this to a depth of a few million reads per sample, i.e., much more reads than there were initial cDNA molecules, then one is bound to see most transcript molecules several times and some individual cDNA molecules, which fared especially well in the PCR, can easily produce hundreds of reads, all showing fragments of the very same mRNA molecule.
Hence, in a setting with very low amounts of starting RNA (typically, below ~1 µg of total RNA per sample), it is quite possible to see a huge difference, say hundreds of reads in one sample and zero reads for the same gene in all other samples, and in reality, this reflects a difference of only a single lone mRNA molecule.
See also our paper on this issue:
Brenencke, Anders, Kim et al., Accounting for technical noise in single-cell RNA-seq experiments
Nature Methods, 10, 1093–1095 (2013)
doi:10.1038/nmeth.2645
In such cases, it is not too uncommon that one only gets maybe around a million mRNA molecules per sample, and after reverse transcription, this is down to possibly less than a hundred thousand cDNA molecules, which then go through many rounds of PCR. If one then sequences this to a depth of a few million reads per sample, i.e., much more reads than there were initial cDNA molecules, then one is bound to see most transcript molecules several times and some individual cDNA molecules, which fared especially well in the PCR, can easily produce hundreds of reads, all showing fragments of the very same mRNA molecule.
Hence, in a setting with very low amounts of starting RNA (typically, below ~1 µg of total RNA per sample), it is quite possible to see a huge difference, say hundreds of reads in one sample and zero reads for the same gene in all other samples, and in reality, this reflects a difference of only a single lone mRNA molecule.
See also our paper on this issue:
Brenencke, Anders, Kim et al., Accounting for technical noise in single-cell RNA-seq experiments
Nature Methods, 10, 1093–1095 (2013)
doi:10.1038/nmeth.2645
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