The benchmark for expression analysis methods compared with reference-based RNA-seq analysis using RNA-seq data derived from WT and GRIN2B edited iPSC-derived cortical neurons. (A) Comparison of different expression analyses. A Venn diagram comparing the de novo transcripts (duplicate counts on a predicted ORF basis), which had potential off-target sites with up to 8 nt MMs, was detected by the dTPM and dDE approaches. dTPM indicates that the expression is decreased based on the ratio of TPM counts between WT and edited samples (left callout). dDE means the expression is reduced based on DEG analysis between WT and edited samples (right callout). (B) Comparison of de novo transcriptome assembly- and reference-based analysis on the deleterious off-target detection. A Venn diagram comparing the off-target genes identified from de novo transcriptome analysis [dTPM (t = 0.4) and dDE (α = 0.001) approaches] and reference-based RNA-seq analysis (CRISPRroots, “RISK: CRITICAL”). (C) Genomic sequence map of off-target located outside of GALR2 mRNA. The sequence is a part of the hg38 reference genome with annotations of GALR2 mRNA (XM_047436984.1) and the de novo transcript (TRINITY_DN86617_c0_g1_i1) and an off-target site with three MMs compared to the on-target sequence. (D) Summary of deleterious off-target sites detected by dTPM and reference-based RNA-seq analysis (CRISPRroots, “RISK: CRITICAL”). The counts of off-target sites are annotated with genes and classified by MM number related to the on-target sequence. The brackets indicate the number of transcripts, including those with and without identified gene annotations.
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