Nextseq system

From 15 randomly selected E. coli strains, nucleic acid isolation was performed using the Higher Purity^TM Bacterial Genomic DNA Isolation Kit (CanvaxBiotech; Valladolid, Spain). DNA concentration was measured with the Qubit ds-DNA HS Assay Kit (Invitrogen; Waltham, MA, USA). Genomic libraries for sequencing were prepared using the Nextera XT DNA Prep Kit (Illumina; San Diego, CA, USA) and purified on AMPure XP magnetic beads (Beckman Coulter Life Sciences; Brea, CA, USA). The quality of the libraries was checked by electrophoresis with the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Paired-end sequencing with 2 × 150 bp reads was carried out on a NextSeq system (Illumina, San Diego, CA, USA).

We used the PBAT capture protocol combining PBAT and hybridization with an RNA probe library (capture), as described in detail in Meir Z. et al. Nature Genetics 2020, 10.1038/s41588-020-0645-y. Bisulfite conversion was performed with the EZ DNA Methylation-Lightning Kit (Zymo Research cat#D5031) following the manufacturer’s instructions. Converted DNA samples were subjected to an End repair reaction containing End repair mix and buffer (NEB cat#E6050) and 0.2–150 ng DNA. DNA was purified using 2.5× SPRI Agencourt AMPure XP beads (Beckman Coulter cat#A63881). The eluted product was next subjected to an A-tail reaction including 10 mM dATPs and Klenow Fragment 3′→5′ exo- (NEB cat#M0212). DNA was purified with 2.5× SPRI beads. The clean DNA was tagged with an index oligo adapter in a ligation reaction using the Quick ligase Kit (NEB cat#M2200). Tagged products were then cleaned using 1.3× SPRI beads, and amplified for library preparation with 14 PCR cycles using the KAPA HiFi HotStart Ready Mix kit (Kapa Biosystems cat#KK2601), following the manufacturer’s protocol. The reaction mix was then cleaned with 0.7× beads. Final libraries were pooled and sequenced on an Illumina NextSeq system using the 150-bp high-output sequencing kit.

Fresh‐frozen tumor tissue samples from the surgical resections (obtained before chemotherapy treatment) were collected from the institutional biobank. After quality control tests were performed, the Illumina NextSeq system was used to sequence the whole mRNA expression of these samples.
Based on these sequencing data, we used R language to calculate the Spearman correlation between gene expression and EMVI. The threshold of correlation significance was set at |Spearman correlation coefficient| >0.7 and p < 0.05. The R language heatmap package was used to draw the heatmap of EMVI‐related genes in GC.

Equal volumes of each of the nine DNA extracts were pooled together and used to prepare a single shotgun library using the Nextera XT DNA library kit (Illumina, San Diego, USA). ESS was performed using a NextSeq® 500/550 v2 high output 300 cycle kit on an Illumina NextSeq system located in the Trace and Environmental DNA (TrEnD) Laboratory at Curtin University. Sequences with an average Q score ≤25 that contained no ambiguous nucleotides and were 151 base pairs (bp) in size (corresponding to the length of a DNA fragment sequenced uni-directionally), were compared to the National Center for Biotechnology Information (NCBI) nucleotide database using BLASTN on the Magnus Cray XC40 system located in the Pawsey Supercomputing Centre at Technology Park in west Australia. Assignment of sequences to taxa at a particular taxonomic level was assessed using the software MEGAN 5.11.3^{70 (link)} using a Min Score of 100 and a Top Percent of 10 under the LCA Parameters.

DNA libraries for whole-genome sequencing (WGS) were prepared with the Nextera (XT) kit from Illumina (San Diego, USA) according to the manufacturer’s instructions [21 (link)]. Pooled DNA libraries were then loaded into NextSeq Reagent cartridges for sequencing on a NextSeq system (Illumina, San Diego, CA, USA). Resulting sequencing reads were submitted to the European Nucleotide Archive under the project accession number PRJEB50767 and subsequently mapped to the H37Rv reference genome (GenBank ID: NC_000962.3) by Burrows–Wheeler alignment (BWA) tool aiming for a minimum of 50-fold average genome-wide coverage [22 (link),23 (link)]. We considered single nucleotide polymorphisms (SNPs) with at least 4 reads in both forward and reverse orientation, 4 reads calling the allele with at least a Phred score of 30, and 75% allele frequency for a concatenated sequence alignment. SNP positions that had reliable base call (as described above) in at least 95% of the strains were concatenated to a sequence alignment. SNPs from repetitive regions were excluded, including those which occurred within a window of 12 base pairs in neighboring strains [23 (link)].
A web tool was used for detecting isolates that harbored more than one phylogenetic lineage (i.e., mixed infections or laboratory contaminations) [24 (link)]. These samples/isolates were not considered in subsequent analysis.

The SMARTer Stranded Total RNA-Seq kit (Pico Input Mammalian) (ClonTech, Mountain View, USA) was used for direct construction of libraries starting with 10 ng of RNA. The workflow used with this kit incorporates a proprietary technology that depletes ribosomal cDNA using probes specific to mammalian rRNA and some mitochondrial RNA. Sequencing was performed on the NextSeq system (Illumina, San Diego, CA, USA) using the NextSeq 500/550 high output kit v2 (catalog no. FC-404-2002; Illumina). Single-read sequencing was utilized with a read length of 150 nucleotides, resulting in the generation of approximatively 125 million reads per sample.

For SARS-CoV-2 genome sequencing, 200 μL of supernatant containing virus was collected and added to 600 μL TRK lysis buffer plus beta-mercaptoethanol (BME) according to the E.Z.N.A. Total RNA kit I (Omega Bio-Tek). Total RNA was extracted according to the manufacturer’s protocol and eluted into RNase/DNase-free H₂O and used for library construction. rRNA was removed from total RNA by Ribo-Zero depletion (Illumina). Indexed sequencing libraries were prepared using TruSeq RNA library preparation kit (Illumina), pooled, and then sequenced using the Illumina NextSeq system. The raw sequence data were analyzed using the LoFreq pipeline to call the mutations in the entire virus genome (42 (link)). In brief, Illumina sequencing fastq data were aligned by BWA with the SARS-CoV-2 reference genome sequence (GenBank accession no. NC_045512) after indexing to generate the aligned SAM and BAM files. The read group was added by Picard after the aligned BAM file sorting and indexing with SAMtools, and then duplicates were removed by Picard MarkDuplicates. Local realignment was achieved by GATK3, and then variants were called by LoFreq to generate the mutant report file.