https://journals.asm.org/doi/10.1128/mra.00119-22#tab1

Next-Generation Sequencing and FASTA Dataset Processing
To process next generation sequencing datasets, we employed our pipeline (SARS-CoV-2_freebayes) consisting of a bash/UNIX script that runs several programs in sequential order. We processed imputed list of SRA accessions with sra-tools7, generating compressed FASTQ files per sequencing, automatically trimmed with fastp tool (Chen et al., 2018). Then, we aligned each trimmed fastq file against a provided reference genome (Wuhan-Hu-1, GenBank Accession: MN908947.3) using Minimap2 splice-aware aligner in preset mode -ax sr (Li, 2018). We sorted and indexed the resulting BAM files by using Samtools (Li et al., 2009) and performed variant calling on every sorted BAM file, obtaining major frequency viral variants per genome in VCF format using the Freebayes variant calling program, as frequency-based pooled caller (−F 0.49)8 (Garrison and Marth, 2012). Then, we used Jacquard program9 in the python environment (Sanner, 1999) to merge every VCF file containing variants associated to each bam file into a single VCF file, containing aggregated variants from all genomes. In the resulting merged VCF file, we recalculated viral frequencies using several UNIX tools (Kernighan and Morgan, 1982), in combination with vcflib10. We used the variants per genome logfile “logfile_variants_SRA_freebayes” to construct Figure 1B using GraphPad Prism 8 software11. We processed GISAID FASTA genomes in a similar manner. We preprocess a single GISAID genome collection with SeqKit (Shen et al., 2016) to decompose a single FASTA file into individual FASTA files, each file containing a single genome. Then, we aligned every FASTA genome against SARS-CoV-2 reference genome (NC_045512.2) using Minimap2 aligner with preset -ax asm5 (Li, 2018) and performed variant calling on each BAM file using Freebayes variant caller with –min-alternate-count 1 (C 1) option (see text footnote 8), outputting variants in VCF format. With these operations, we obtained major frequency viral variants in VCF format from each FASTA genome. Then, we aggregated variants into a single VCF file, as described with Jacquard. We constructed Figure 1B graph by using variants per genome logfile, reported in the output file “logfile_variants_GISAID_freebayes” and imputed into the GraphPad Prism 8 software. We filtered out highly homoplasic sites from merged variant calls, as already reported to be frequent in SARS-CoV-2 sequencing see: https://virological.org/t/issues-with-sars-cov-2-sequencing-data/473. All these computational analyses are described here: https://github.com/cfarkas/SARS-CoV-2-freebayes (case examples I and II, respectively).