Selection of RNA-seq library preparation protocol for a comprehensive transcriptome analysis in T acute lymphoblastic leukemia (T-ALL)

The aim of this study was to compare two different library preparation protocols: Illumina TruSeq Stranded mRNA (referred to as PA) and TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat (RD) each with two different variants of the fragmentation step: standard fragmentation conditions (94?C for 8 minutes; further described as P1) and modified conditions (90?C for 2 minutes; further described as P2). The modification was expected to produce longer fragments. Each of the 5 tested T-ALL samples was sequenced using a combination of 4 different protocols: PA_P1, RD_P1, PA_P2, RD_P2 (for PA_P2 we performed two technical replicates). All 25 libraries were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 150M reads/sample. High number of reads obtained for each of the samples allowed us to carry out a comprehensive comparison using various analysis methods aimed at identifying differentially expressed genes, alternative splicing events, gene fusions, somatic mutations and indels.