Novoseq platform

All tumor tissues were microscopically evaluated and assessed by a pathologist for tumor content. Genomic DNA was isolated from snap frozen tumor tissue and from matched blood prior to whole exome sequencing. Briefly, hybrid selection was performed with the Human All Exon kit SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA, USA) version 6 on the Illumina NovoSeq platform as paired-end 150-base pair reads. Alignment of read pairs were performed using Burrows-Wheeler Aligner (BWA MEM, http://bio-bwa.sourceforge.net/) to the human reference genome NCBI GRC Build 37 (hg19)^{30 (link)}. Using Picard (http://broadinstitute.github.io/picard/), we marked the optical duplicates prior to base score recalibration with GATK version 4.1.4 for post-alignment data processing^{31 (link)}. To identify somatic variants from the normal and tumor BAM files, we used Mutect2 followed by annotating and prioritizing using VEP^{32 (link)}.

Briefly, 30–50 male or female flies per replicate were crushed in 500 μl homogenization medium (0.1 M Tris, 0.1 M EDTA,1%SDS, 0.5% diethylpyrocarbonate) and incubated at 65 °C for 30 min. The DNA was precipitated using 100 μl of 8 M potassium acetate and Isopropanol^{61 (link)}. The extracted gDNA is amplified using vgsc specific primers followed by adapter primers and run on Illumina NOVOseq platform. Data were analyzed using CRISPResso2 and CrispRVariants pipelines.
vgsc forward: 5′ACACTCTTTCCCTACACGACGCTCTTCCGATCTagcttcatgatcgtgttcc 3′
vgsc reverse: 5′GACTGGAGTTCAGACGTGTGCTCTTCCGATCTgccatggttagaggcgataagtc 3′.

A total of 20–40 mg leaf tissue was weighed out and pulverized using a SPEX^® sample prep tissue homogenizer (SPEX Inc, Metuchen, NJ, United States). DNA was extracted using CTAB and isopropanol (Doyle and Doyle, 1987 ) and cleaned using a 2x ratio of AMPure XP beads (Beckman Coulter, Brea, CA, United States). DNA libraries were prepared using NEB Next Ultra II Library Prep Kits according to the manufacturer’s protocol (with half volume reactions) and with NEBNext Multiplex Oligos for Illumina (New England Biolabs, Ipswich, MA, United States) amplified with 9–11 PCR cycles (fresh samples) or 11–15 PCR cycles (herbarium samples). Yield and fragment size distribution were estimated using a Quantus fluorometer (Promega, Madison, WI, United States) and a 4200 TapeStation system (Agilent Technologies, Santa Clara, CA, United States) respectively. Sequencing of DNA libraries was carried out on an Illumina NovoSeq platform with a paired end 150 bp configuration, by GeneWiz^® (South Plainfield, NJ, United States).

WES data for tumor sample generated from CD138+ cells collected after the second infusion. Peripheral blood mononuclear cells were used as germline control. WES libraries generated using Twist Bioscience Human Core Exome Kit and sequenced as 75 bp paired-end reads with Illumina Novoseq platform. The average sequence coverage for targeted regions was 110× for tumor sample and 602× for germline DNA. We aligned paired-end reads using BWA-mem (v0.7.17-r1188)²⁷ to GRCh38. We followed GATK (v4.0.11) best practice to mark duplicated reads with MarkDuplicates function and base quality score recalibration with ApplyBQSR²⁸ . Mutect2²⁹ was used to call mutations. Only mutation calls with at least 10× coverage for tumor and germline samples and passed FilterMutectCalls function were annoted using Variant Effect Predictor from Ensembl (v100). Allele-specific copy number calls as well as ploidy and purity of the sample were analyzed using FACETS (v0.6.1) (Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing)^{30 (link)}.

F. filiformis strains collected in the study are shown in Table 1 and Table 2. In total, 232 strains of F. filiformis strains were collected in this study, including 157 cultivars and 75 wild strains. To obtain a comprehensive understanding of the genetic variation present within the F. filiformis, we selected cultivars from various countries, although a significant proportion of the strains were sourced from China. The mycelia were grown in solid Potato dextrose agar (PDA) medium at 25 °C until they reached full growth. Genomic DNA was extracted from mycelia using the CTAB method [27 ]. A Nanodrop and 1.0% agarose gel electrophoresis were used to assess the concentration and integrity of the DNA solution. Whole-genome sequencing libraries were prepared using NexteraXT reagents (Illumina). The Illumina Novoseq platform from Novogene was then used for sequencing the DNA samples. Briefly, approximately 2 μg of DNA from each sample was used for fragmentation by Biorupter (high power: (15 s, on/90 s, off), six cycles) and end preparation by NEXT flex TM End-Repair. After PCR amplification (10 cycles), the library was purified using AMPure beads. Qubit was used to evaluate the quality and quantity of each library. The sequencing statistics of the samples are summarized in Table 1.

Total RNA was extracted from tumor, matched normal tissue, and cell lines using the RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). The integrity of RNA was determined by electrophoresis using the 2100 Bioanalyzer (Agilent Technologies, USA). Whole transcriptome sequencing of cell lines was performed on the Illumina NovoSeq platform (Novogene, Singapore) using the standard Illumina RNA-seq protocol. The reads were aligned to the human genome hg19 RefSeq reference transcriptome by STAR^{33 (link)}. Transcript abundance estimation was performed using RSEM³⁴ . Differentially expressed genes were identified using DESeq2^{35 (link)}. For each gene, read counts were represented as “transcripts per million” (TPM) and were normalized for both sequencing depth and gene length. For tumor and matched normal tissue transcriptomic analysis was performed using the Illumina Ampliseq Transcriptome Human Gene Expression Panel (Illumina, San Diego, CA, US) following macrodissection of a representative section from formalin-fixed paraffin-embedded samples. Gene set enrichment analysis (GSEA) was performed using the Molecular Signatures Database (MSigDB) Hallmark gene set^{36 (link)}. A gene set is significantly enriched if its Normalized Enrichment Score (NES) has a False Discovery Rate (FDR) q-value below 0.05.

In brief, DNA was isolated using the Magnetic Stool DNA Kit (Tiangen Biotech, China) in accordance with the manufacturer’s protocols, which were quantified by Qubit® dsDNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, USA). The shotgun metagenomic sequencing was performed on the Illumina NovoSeq platform, generating on average 20 million reads (~6 Gb) per sample. High-quality reads were screened by trimming low-quality (Q-Score <20) nucleotides and potential human contaminants were removed using Bowtie2 (version 2.2.4). Metagenomics taxonomical annotation was performed using DIAMOND (V0.9.9.110) and used a library of NCBI blast to provide pan-microbial (bacterial, archaeal, viral and eukaryotic) quantification at the species level. All samples were compared and analyzed at the species level by relative abundance. The shotgun metagenomic DNA sequencing data from fecal samples were obtained from Beijing Novogene Biotechnology Co., Ltd, China.