Nextseq 500 550 mid output kit v2

The quality of purified RNA was assessed using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, United States) and the Agilent RNA 6000 Pico Reagents Kit (Agilent Technologies). Ribosomal RNA was removed using the QIAseq FastSelect RNA Removal Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA from each sample was prepared for sequencing using the QIAseq Stranded mRNA Library Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Normalized libraries were pooled and sequenced using a NextSeq 500/550 Mid OutputKit v2.5 (Illumina, San Diego, CA, USA) with 150 cycles (2 × 75 bp paired-end) on a NextSeq 550 platform, generating a depth of 5–16 million paired-end reads for each sample. Reads were trimmed and filtered using Trimmomatic [49 (link)], and reads were aligned to the E. histolytica transcriptome (AmoebaDB, Release 61, 15 Dec 2022) using RSEM [50 (link)] and Bowtie2 [51 (link)] software. Differential expression was tested using DEseq2 to normalize the raw data [52 (link)].

Whole-exome sequencing (WES) using the TruSeq DNA Exome Kit, the NextSeq 500/550 Mid Output Kit v2.5, and a NextSeq 500 sequencing device (all Illumina, CA, USA) was done in all three cases. Input material was 400 ng of DNA obtained from the peripheral blood (for germline exome) and formalin-fixed, paraffin-embedded (FFPE) tumor sample with ≥20% cancer cell count measured in the surface area of tissue slides for somatic exome. WES was done with high coverage where at least 90% of targeted regions were covered 20 times.

Campylobacter strains grown on ColbA for 24 h under microaerobic atmosphere at 42 °C were harvested and DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, USA). The quality of the DNA was evaluated by spectral analysis (NanoDrop Spectrophotometer, Thermo Fisher Scientific, USA) and the concentration was fluorimetrically quantified by Qubit 3.0 Fluorometer (dsDNA HS Assay Kit 0.2–100 ng; Thermo Fisher Scientific, USA). DNA libraries were prepared using the Nextera XT DNA Library Prep kit or the Nextera DNA Flex Library Prep Kit according to the manufacturer’s instructions (Illumina, San Diego, USA). Quality of the libraries was assessed by gel analysis or on a fragment analyser 3408 (Advanced Analytical Technologies Inc., USA). Paired-end sequencing was performed on the Illumina MiSeq (2×301 cycles) or the NextSeq (2×151 cycles) platform using the MiSeq v3 (600 cycles) reagent kit or the NextSeq 500/550 Mid Output kit v2.5 (300 cycles), respectively. The sequences were published within the BioProject No. PRJNA595957, BioSample No. SAMN13577876-SAMN13577920, SRA accession No. SRR10698060-SRR10698104 at NCBI sequence read archive (SRA). New MLST alleles and MLST-ST types were uploaded to PubMLST.

DNA was extracted on a QIAsymphony SP system with the QIAsymphony DSP virus/pathogen kit (Qiagen) according to the manufacturer’s instructions. The DNA concentration was quantified using the Quant-IT double-stranded DNA (dsDNA) high-sensitivity kit (Thermo Fisher Scientific).
Sequencing libraries were prepared from the pure A. baumannii DNA extract samples using the Nextera XT library preparation kit (Illumina Inc.) with slight modifications. One-half of the volume was used for tagmentation reagents, amplification reagents, and input DNA. Library cleanup was performed using the AxyPrep MAG PCR cleanup kit (Corning Inc., NY, USA), and libraries were pooled manually and sequenced on a NextSeq 550 platform with the NextSeq 500/550 midoutput kit v2.5 (300 cycles) (Illumina Inc.). Genome mutations were identified using breseq (39 (link)).

Genomic DNA was extracted using the MN NucleoSpin^®Microbial DNA (Machery-Nagel GmbH and Co.KG, Duren, Germany) kit following the manufacturer’s instructions. The quantification of extracted genomic DNA was determined using a DeNovix DS-11 + Spectrophotometer. WGS was conducted at a high-throughput sequencing facility at SA Pathology in South Australia. Sequencing libraries were prepared using the Nextera XT DNA library preparation kit (Illumina Inc., San Diego, Ca, USA), with modifications of the kit’s protocol. Briefly, genomic DNA was fragmented, followed by the amplification of Nextera XT indices (Illumina Inc., San Diego, CA, USA) to the DNA fragments using a low-cycle PCR reaction. The amplicon library was then purified, and normalised manually. Whole-genome sequencing (WGS) was performed on the Illumina NextSeq 550 platform with NextSeq 500/550 Mid-Output kit v2.5 (300 cycles) (Illumina Inc., San Diego, Ca, USA).

After RiboGreen quantification and quality control by Agilent BioAnalyzer, 11–259 ng of total RNA were prepared using the Immunoverse TCR-HS α/δ/β/γ Kit, for Illumina (ArcherDX catalog # DB0219) according to the manufacturer’s instructions. Briefly, cDNA was synthesized using TCR-specific priming for reverse transcription. Molecular barcode adapters were ligated to cDNA fragments and multiplex PCR with primers targeting the CDR3 sequence of interest was used for enrichment and library preparation. Barcoded samples were pooled equimolar and sequenced on a MiSeq, NextSeq 500, or NovaSeq 6000 in a PE150 run, using the MiSeq Reagent Kit v3 (300 cycles), NextSeq 500/550 Mid Output Kit v2.5 (300 cycles), or NovaSeq 6000 S3 Reagent Kit (300 Cycles) (Illumina). Each sample yielded on average 3.2 M reads and fastq files were uploaded to the Archer Analysis bioinformatics suite for processing.
Archer delivered trimmed and deduplicated fastqs, which were then used to quantify TCR diversity for the 4 TCRs analyzed by the platform (α, β, γ, and δ). The MiXCR pipeline was used to process, align, assemble, and export clones for each TCR type separately⁶⁹ . Next, the VDJtools pipeline⁷⁰ was utilized to calculate basic statistics including TCR diversity. Differences in TCR diversity were compared by two-sided t-test between each response group (PR, SD, or PD) vs. others.