Dnbseq g400 system

The genomic DNA was isolated from the peripheral blood of the patient using the NucleoSpin Blood mini kit for DNA (Macherey–Nagel, Germany) and used for whole-exome sequencing (WES). Collection of raw sequencing data, alignment with the reference human genome, variant calling, and annotations were performed by WES service provider, Genoks, Turkey. Briefly, WES was done using the Human Comprehensive Exome panel (Twist Bioscience, USA), followed by paired-end sequencing performed on DNBSEQ-G400 System (MGI Tech, China), generating 150 base-paired ends. BCL2Fastq2 v2.20 was used for FASTQ generation. BWA-MEM software was used for the alignment with the human genome reference sequence (GRCh37). Genome Analysis Toolkit and SnpEff were used for downstream processing. The gnomAD 2.1.1 (https://gnomad.broadinstitute.org/) and 1000 Genomes Project (http://grch37.ensembl.org/) databases were used to obtain allele frequencies.

Extracted viral RNA was reverse transcribed and tagged with index adaptors using the NEBNext Ultra II RNA library prep kit for Illumina (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. The resulting cDNA libraries were verified using the MultiNA system (Shimadzu, Kyoto, Japan) and quantified using a Quantus fluorometer (Promega, Madison, WI, USA). Indexed libraries were then converted and sequenced (150-bp paired-end reads) using the DNBSEQ-G400 system (MGI Tech, Shenzhen, China; operated by Genewiz, South Plainfield, NJ, USA). After sequencing, reads with the same index sequences were grouped. Sequence reads were trimmed by Ktrim (42 (link)) and mapped onto the viral genomes of parental strains using Minimap2 (43 (link)). The consensus sequences of the mapped reads were obtained using ConsensusFixer (A. Töpfer [https://github.com/cbg-ethz/consensusfixer]).