Miniseq mid output kit 300 cycles

Genomic DNA was isolated from overnight LB cultures of LT2 and MTs strains using the GeneJET genomic DNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA), after which 150 bp paired-end libraries were prepared using the Nextera DNA Flex library prep kit (Illumina, San Diego, CA, USA) and Nextera DNA CD indexes (set of 24 indexes) (Illumina). Sequencing was carried out with an Illumina MiniSeq sequencer using a MiniSeq Mid Output Kit (300-cycles) (Illumina, San Diego, CA, USA) and analyzed with QIAGEN CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark, https://digitalinsights.qiagen.com/). The sequencing reads of our LT2 wild-type strain were trimmed and mapped to the NCBI reference genome (LT2, NCBI accession number: NC_003197.2) to generate a reference consensus sequence of our own wild-type LT2 strain. The sequencing reads of the mutant strains were then trimmed and mapped to this newly made reference LT2 genome and analyzed for single-nucleotide polymorphisms (SNPs, via basic variant detection command), indels (InDels, via InDels, and structural variants command), and structural variants (SVs, via InDels, and structural variants command). All mutations detected by WGS were verified by Sanger sequencing.

The pooled libraries were mixed with the PhiX Control v3 Library (final 10%; Cat# FC-110-3001, Illumina, San Diego, CA, USA), diluted to 1 nM, and then subjected to denaturation and neutralization processes. Subsequently, the libraries were diluted further to 1.4 pM and then applied for an NGS run using a MiniSeq Mid Output Kit (300-cycles) (Cat#FC-420-1004; Illumina) in the MiniSeq System (Illumina). The sequencing was performed with the paired-end reads of 150 bases. The cluster density was 312 K/mm². In addition, the passing filter rate of over Q30 for the clusters was 88.39%, and the data yield was 5.57 G bases with 32.2 M paired-end reads. Overall, the NGS run was considered capable of obtaining high-quality data. Subsequently, FASTQ files were exported following the performance of further bioinformatics analyses.

Genomic DNA was isolated on day 4 after electroporation unless otherwise stated. Two hundred-base pair PCR amplicons (KAPA HiFi HotStart 2 × Readymix; Roche) of the B2M and CIITA loci-containing DNA adapters were generated using primer pairs and adapters published by Gaudelli et al. [42 (link)]. The PCR products were purified using AMPure beads (AMPure XP, Beckman Coulter Genomics) and the size was checked on a 1.5% agarose gel. In a second PCR (KAPA HiFi HotStart 2 × Readymix; Roche), dual indices were added using primers binding to the previously attached adapters (Additional file 2: Table S5). The PCR products were purified using AMPure beads (AMPure XP, Beckman Coulter Genomics), and the size was checked on a 1.5% agarose gel. The concentration was measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific) and adjusted to 4 µM. Twenty microliters (4 µM) of each samples were united to a library and diluted to a 1 µM concentration. Five microliters of Library (1 µM) was denatured using 5 µL of 0.1 N NaOH (Sigma) and diluted to a loading concentration of 1.4 pM. The library was spiked with 20% of diluted and denatured 1.4 pM PhiX control (Illumina), and 500 µL of the final sample was sequenced 2 × 150 cycles using the MiniSeq Mid Output Kit (300-cycles) (Illumina) on an Illumina MiniSeq instrument.