Fig. 3

Illumina size standards allow measurement of sequencer-specific size biases. a Design of REcount-based Illumina size standard constructs. Each standard construct contains a normalization barcode, as well as a barcode associated with a variable size standard that can be liberated by MlyI digestion and directly sequenced. b Raw abundance data for all 30 size standards and normalization barcodes from a MiSeq run. c Run-to-run variability of multiple MiSeq runs (n = 6 flow cells). d Size bias profiles of the iSeq (n = 1 flow cell), MiSeq (n = 6 flow cells), NextSeq (n = 4 flow cells), and NovaSeq (n = 4 flow cells, 4 lanes) sequencers. Note: Size bias data for other Illumina instruments is shown in Additional file 1: Figure S5. e Size bias profiles of the same library either clustered on the MiSeq immediately after denaturation or clustered after freezing and thawing the denatured library. Error bars are ± s.e.m