IVT-seq reveals extreme bias in RNA sequencing

Table 2 Fold-change differences in within-transcript coverage by library type

	Number of IVT-transcripts with fold-change differences:
	>2	>10	>100
rRNA-depleted	713 (74.0%)	110 (11.4%)	17 (1.7%)
PolyA selection	678 (70.4%)	163 (16.9%)	7 (0.7%)
No selection	400 (41.5%)	31 (3.2%)	3 (0.3%)
Plasmid	189 (19.6%)	14 (1.5%)	3 (0.3%)
Simulated	0	0	0
QM-simulated	0	0	0

The plasmid data provides a measure of bias from library preparation/sequencing, while the no selection data accounts for potential artifacts from the in vitro transcription (IVT) step. To calculate the percentage of transcripts affected by bias due specifically to library preparation and sequencing, but not sequence or in vitro transcription artifacts, we performed the following calculation: rRNA depletion% - no selection% + plasmid%. So we found 74% – 41.5% + 19.6% = 52.1% of transcripts in the rRNA-depleted data have greater than two-fold difference in coverage, and 11.4% – 3.2% + 1.5% = 9.7% have greater than 10-fold difference in coverage. QM, quantity matched.

ISSN: 1474-760X