Fig. 9

Overview of iVar pipeline. iVar was used to construct two pipelines for calling intrahost single-nucleotide variants (iSNVs) from samples with and without a known reference sequence. The nodes in the chart are colored based on the usage of iVar, bwa, and SAMtools at each step. For samples with a known reference sequence, the primer sequences are trimmed from the sequenced reads, followed by quality trimming. A consensus sequence for the sample is called by merging the aligned BAM files from each replicate. The primer sequences are then aligned to this consensus sequence and mismatches are identified by iVar after performing variant calling on the aligned primers. The reads corresponding to the mismatched primers are removed from the aligned BAM file of each replicate to ensure that any bias introduced in the iSNV frequencies is removed. The iSNVs are then called for each replicate, individually, with a minimum frequency threshold of 3% and an intersection of the iSNVs across all the replicates are considered to be the “true” iSNVs. For samples with an unknown reference sequence, the iSNVs cannot be called directly using a reference sequence. In this case, after generating the consensus sequence, reads from each replicate are aligned back to this consensus sequence and these realigned BAM files are used for the same subsequent steps as in the case of samples with a known reference sequence