Miniseq high output reagent kit

RNA-seq was performed as previously described^{29 (link)}. Sequencing libraries were prepared using the QuantSeq 30 mRNA-seq LibraryPrep Kit FWD for Illumina (Lexogen, Inc.). After removal of the RNA template, a second strand was synthesized using random primers. The double-stranded cDNA library was purified using magnetic beads after washing, and the library was amplified by PCR using primers containing adapters and i7 index sequences. Libraries were purified with magnetic beads, quantified using an Affluorometer (Qubit 4, Thermo Fisher Scientific), and pooled to equimolar concentrations. Sequencing was performed on a Mini Seq system (Illumina, San Diego, CA, USA) with 75 cycles using the Mini Seq High Output Reagent Kit. After sequencing, sequence quality was examined using FastQC.
Gene expression difference analysis and pathway analysis were then performed by iDEP. Gene expression difference analysis was performed using DEseq2 [false discovery rate (FDR) cutoff: 0.1, minimum fold change: 2]. Pathway analysis was performed on the GO Biological Process gene set using Generally Applicable Gene Set Enrichment for pathway analysis (GAGE; FDR cutoff:0.10).

RNA was extracted from the supernatant of cells infected with Candid #1 (P2, P4, P6, P8, and P10) using the QIAamp Viral RNA Mini Kit (Qiagen), according to the manufacturer’s instructions. The regions including Favipiravir-resistant mutations in the L and S segment were amplified by RT-PCR as described above. After purification using agarose gel electrophoresis, libraries of PCR products were prepared with the NEBNext Ultra II FS DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). Sequence analysis was performed using the MiniSeq instrument (Illumina, San Diego, CA, USA) with the MiniSeq High Output Reagent Kit (Illumina). The obtained reads were trimmed and mapped to the reference JUNV Candid #1 sequence using the CLC Genomics Workbench software (Qiagen). The proportion of mutations at each time point was calculated using the number of mapped reads.

Targeted sequencing was performed using AmpliSeq for Illumina Myeloid Panel (Illumina, San Diego, CA, USA) and a custom-designed panel to detect mutations in 68 genes and fusions of 29 driver genes (S1 Table). As a template, 10 ng DNA (for mutations) or cDNA synthesized from 10 ng RNA (for fusions) was used to amplify the target genes. AmpliSeq Library Plus for Illumina (Illumina) was used to generate libraries. The size of the fragment libraries was determined using the 2100 Bioanalyzer. The libraries were analyzed using the MiniSeq High Output Reagent Kit (300 -cycles) with the MiniSeq (Illumina) platform in accordance with the manufacturer’s instructions.

From each sorted population, as well as ancestor sample, genomic DNA was extract using PureLink genomic DNA mini kit and used as templates for PCR to amplify specifically the sgRNAs in the population. PCR reaction was conducted using 2X KAPA HiFi HotStart ReadyMix, 10 µM of each primer and 20 ng of genomic DNA extracted from the samples in a 50 µl total volume reaction, for 20 cycles, Tm = 58°C. We used shifted primers to increase library complexity. PCR products were purified with SPRI-beads using left-side size selection protocol in which the PCR product and beads were mixed at 1:1.5 ratio. The barcoding PCR and final PCR clean-up was done as described for the genomic tRNA library preparation. Samples were pooled and sequenced using a 75 bp single read output run using MiniSeq high output reagent kit (Ilumina; FC-420–1001).
Reads were trimmed using Cutadapt and then clustered into unique sequences using vsearch (Rognes et al., 2016 (link)). Each unique read was then aligned to the matched sgRNA sequence, allowing up to two mismatches. Finally, we stored all sgRNA types and their frequency in each sample.