Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification.

Gohl DM, Magli A, Garbe J, Becker A, Johnson DM, Anderson S, Auch B, Billstein B, Froehling E, McDevitt SL, Beckman KB.

Genome Biol. 2019 Apr 29;20(1):85. doi:10.1186/s13059-019-1691-6

Sequencing templates already containing full Illumina adapters are cleaved from plasmids with a type IIS enzyme that generates blunt ends. Comparison between fixed-length and variable-length payloads allowed to study the size bias in Illumina sequencers. NextSeq > iSeq > MiSeq > NovaSeq. A freeze-thaw cycle reduced the prevalence of the smallest templates (~150 bp).