Nextseq 500 550

The RNA-seq protocol carried out in this manuscript was previously described [28 (link),29 (link)]. In summary, a next-generation sequencing run for whole transcriptome sequencing was performed using a paired-end 2 × 75 nt library on a NextSeq 500/550 Illumina sequencer and using a high-output Kit v2.5 (150 cycles), which generated an average of 18.9 million reads per sample. Raw data were generated for each of the libraries by the genomic core service at GENyO (Granada, Spain). The RNA-seq data are available at NCBI Short Read Archive (SRA) under accession number PRJNA836366.

RNA-seq was performed as by MARS-seq as described in [55 (link)]. The bulk MARS-seq libraries were sequenced with high-output 75-bp kits (FC-404-2005, Illumina, USA) on NextSeq 500/550 Illumina sequencer.
Processing of raw sequencing data into read counts was performed via the User-friendly Transcriptome Analysis Pipeline (UTAP) [56 (link)]. Reads were trimmed using cutadapt [57 ] and mapped to genome (/shareDB/iGenomes/Mus_musculus/UCSC/mm10/Sequence/STAR_index) using STAR [58 (link)] (default parameters). The pipeline quantifies genes annotated in RefSeq (that have expanded with 1,000 bases toward 5′ edge and 100 bases toward 3′ bases). Counting (UMI counts) was done using HTSeq-count in union mode [59 (link)]. Count normalization was performed using DESeq2 [60 (link)] with the following parameters: betaPrior = True, cooksCutoff = FALSE, and independentFiltering = FALSE.
RNA-seq data are available from the GEO database (accession number GSE171975). All other data that support the findings of this study are available from the corresponding author upon request.

Sequencing was performed in a NextSeq 500/550, using a Mid Output Kit (2 × 150 bp) (Illumina). A depth of 4000x was estimated for targeted panel libraries (clinical samples and reference strains) to detect minor populations of Mtb in co-infections and possible heteroresistance (10 (link)). The depth of WGS libraries (reference strains only) was at least 200x.
The quality of all libraries was evaluated on an Agilent 4200 TapeStation System, and their concentration determined with the Qubit high sensitivity dsDNA assay (Qubit 3.0, Thermo Fisher) before sequencing.

The entire coding regions of fifty-seven genes were included in a hybridisation capture panel (Supplementary File 1_Table 1), based on: recurrence in our WES tumour set; reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database; implicated in cancer^{65 (link)}; or reported in the TCGA or Bueno et al. studies^{4 (link),5 (link)}. Sequencing libraries were prepared from DNA extracted from tumours and normal tissue (whole blood) samples using the SureSelect QXT Target Enrichment System (Agilent, Santa Clara, USA) according to the manufacturer`s protocols. Sequencing was performed on a MiSeq or NextSeq500/550 platform (Illumina) with a mean read depth of 780.6X (all samples). Supplementary File 2_Table 3 contains a list of filtered somatic variants from targeted capture sequencing.

For molecular analysis, 3–10 5–10µm micrometer formalin-fixed paraffin-embedded (FFPE) sections were prepared and tumor tissue was micro-dissected when the tumor content was below 10%. DNA was extracted semi-automated (Maxwell® 16, Promega), and 400ng of input DNA was sonographically sheared (Covaris®) into approximately 200-bp double-stranded fragments. Hereafter, adapters were ligated, and genomic regions of interest were enriched using complementary bait sequences. During this hybrid capture, the selected baits ensure optimal coverage of all relevant genomic regions, including 340 genes in a 1.14 Mb complete genomic territory size. Following the enrichment, the targeted fragments were clonally amplified and sequenced with next-generation sequencing (NextSeq 500/550, Illumina). Point mutations, small insertions and deletions, copy number alterations, and rearrangement/gene fusions were identified with NEO New Oncology’s proprietary computational biology analysis pipeline and analyzed using the NEO diagnosis software.

During August 2021, 180 SARS-CoV-2 positive samples with N1 Cq values less than 30 were randomly selected from a total of 710 saliva isolates collected from Clemson students and employees. These heat-treated positive saliva samples were then sent for sequencing to an external reference lab (Premier Medical Sciences, Greenville SC, USA). RNA was extracted from saliva samples via magnetic beads (Omega) and recovered SARS-CoV-2 RNA quantity was assessed via Codiagnostics Logix smart assay. Samples were processed and sequenced on either an Illumina NovaSeq 6000 or NextSeq500/550 flow cell. Sequences were demultiplexed, assembled, and analyzed in DRAGEN COVID Lineage (Illumina, v.3.5.3). The delta variant was present in 95.6% of sequenced university samples. Between 8/22/2021 and 9/18/2021, it is estimated that the delta variant was present in 98.9% (95% CI: 93.5-99.9%) of SARS-CoV-2 positive samples in SC⁴⁹ .