Miseqdx instrument

NGS analysis was performed for the validation of IDH1 and IDH2 mutations. Genomic DNA was extracted from tumors after macro-dissection using the Gene-Read™ DNA FFPE Kit (Qiagen, Hilden, Germany), in accordance with the manufacturer's protocol. Targeted sequencing for 90 cancer-related genes was performed using the SureSelect targeted panel (Agilent Technologies, Santa Clara, CA, USA), as previously described (15 (link)). The processed libraries were loaded onto the MiSeqDx instrument (Illumina, San Diego, CA, USA), in accordance with the manufacturer's protocol. Sequenced reads were aligned to the human reference genome (GRCh37/hg19) using the BWA-MEM (0.7.15); common germline variants were distinguished from somatic variant candidates in accordance with a previously described method (15 (link)).

According to the manufacturer's protocol, the biopsied TE cells were lysed and the whole genome was amplified using a SurePlex WGA Kit (Illumina). For chromosomal analysis with aCGH, the WGA products and control DNA were labeled with Cy3 and Cy5 fluorophores according to the manufacturer's instructions and hybridized on 24sure + arrays (Illumina). For chromosomal analysis with NGS, the WGA products were used for library construction with the VeriSeq DNA Library. According to the manufacturer's protocol, NGS was performed on a MiSeqDx instrument (Illumina) using MiSeqDx Universal Kit 3v (Illumina). All results were analyzed using BlueFuse Multi analysis software (Illumina) for chromatin loss or gain across all 24 chromosomes.

The data obtained during bioinformatics analysis were subjected to quality control as follows: The raw reads in FASTQ format from the Illumina MiSeq Dx instrument were first filtered for quality using Trimmomatic v0.39 with a Q20 [53 (link)]. Next, data filtered by read quality were aligned to the standard reference Wuhan-Hu-1 comparison genome (MN908947.3) using minimap2 v2.24 [54 (link)]. The primer sequences of the multiplex panel for RT-PCR that did not contain genomic information were removed from the aligned reads. Then, searching (colling) for variants was performed using GATK v4.2.6.1 [55 (link)]; specifically, mapped reads were compared with the comparison genome to identify variants (differences) of the analysed sample relative to the reference genome if more than five were covered. Based on the variants found, the consensus sequence of the SARS-CoV-2 genome was collected in fasta format using the Bsftools program [51 (link)]. Lineages were assigned with Pangolin v.4.0.6 using pango-data v.1.9.