Fig. 1

Global assessment of SCRB-seq’s performance for bulk RNA-seq. a Comparison of read alignment performances between TruSeq and five SCRB-seq datasets: one lymphoblastoid cell line (LCL; generated in-house), and four public datasets from [15, 18]. The no/multiple alignment values are derived from the STAR [35] alignment, and no gene/ambiguous and mapped to genes correspond to the annotation of the reads to the genes by Htseq [49]. b Total number of detected genes in the same LCL RNA samples by SCRB-seq and TruSeq at different detection thresholds (e.g., “Reads > 0” means that a gene is considered detected if it is covered by at least one read). c Evaluation of SCRB-seq’s performance relative to TruSeq using the data downsampled to 1M single-end reads and shown by the total number of identified DE genes and number of “true positive” DE genes. The latter represents a subset of DE genes identified using the full TruSeq 30M paired-end set; the error bars correspond to the variation produced by downsampled replicates (see the “Methods” section). d Assessment of the impact of the number of cycles during PCR pre-amplification of SCRB-seq libraries (downsampled to 1M single-end reads) prepared with BU3 primers. Performances were evaluated through variable quality measures: uniquely mapped reads, level of duplication, rate of MT-rRNA reads, and number of detected genes. e Assessment of the complexity of the libraries (downsampled to 100k single-end reads) obtained with different combinations of RT enzymes and DS cDNA generation procedures at various detection cutoffs (e.g., “Reads > 0” means that a gene is considered detected if it is covered by at least one read). f Read coverage across the gene body for different combinations of RT enzymes and DS cDNA generation procedures. Legend: DS cDNA, double-stranded cDNA; SE, single end; MMH, Maxima Fermentas Minus H Enzyme; SSII, Superscript II enzyme; SSS, second-strand synthesis using Nick translation; PCR, pre-amplification by polymerase chain reaction