Observed bias in amplification-based sequencing.

(A) Single-best mapping method-based quantile-quantile plot demonstrates evidence of over-representation of highly expressed transcripts in amplification-based sequencing compared to single-molecule methods. (B) Distribution of reads across genes by transcript concentration shows decreased SMS coverage of the most highly expressed genes, with those reads going to mid- and low-level expressors. (C) Differences in the distribution of reads lead to increased sensitivity of low-expressing transcripts. (D) Nine of the candidate genes seen above the 0.3 RPKM noise level demonstrated any amplification by RT-PCR, although only HIST1H4C showed high abundance.