Fig. 2.

PASTMUS workflow and bioinformatics pipeline to identify critical residues of proteins. a PASTMUS workflow. sgRNA tiling library screening was performed, followed by amplification of sgRNA barcodes and the targeted gene cDNA for NGS. After mapping to targeted gene CDS and extracting mutation, sequencing reads with out-of-frame mutations were filtered out. The frequency of reads conferring amino acid deletion, combo mutation, and substitution were calculated for libraries before and after screening. Quantitative assessment (fold change) of a.a. deletions and combo mutations and qualitative assessment of a.a. substitutions were performed, respectively. b–f The essential scores of individual amino acids indicating their functional significance were calculated accordingly. The PASTMUS bioinformatics pipeline is as follows: mapping of NGS data with reference sequence (b), applying the filtering rules for NGS data (c), calculating a.a. substitution frequency and setting noise threshold for filtering (d), calculating fold change of a.a. deletion and a.a. combo mutation between libraries before and after screening (e), and computing essential scores for individual a.a. according to quantitative assessment of a.a. deletion and a.a. combo mutation, and qualitative assessment of substitution frequency (f)