Fig 4

The sensitivity of the new approach based on PacBio sequencing and clustering analysis in detection of minor gene isoforms in a mixture containing multiple isoforms. Twenty-two plasmids, representing 22 different P. jirovecii msg isoforms previously cloned [22], were mixed with various concentrations and amplified by PCR for the full-length msg coding region (~3 kb) followed by PacBio sequencing and clustering analysis. The read frequency and concentrations of plasmids (indicated by diamonds) in the mixture are positively correlated. The concentration is the percentage of each plasmid DNA relative to the total amount of plasmid DNA in the mixture. Those 4 msg isoforms, which were not identified, have the lowest concentrations (0.8–1.3 %) in the mixture