Qubit high sensitivity dsdna quantification assay kit

Genomic DNA from chigger pools were extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. DNA concentration and quality were assessed by a Qubit High Sensitivity dsDNA Quantification Assay kit (Invitrogen) and NanoDrop One/One^C Microvolume UV-Vis Spectrophotometer (Thermo Scientific). A quantitative PCR assay (qPCR) targeting the multicopy traD gene was used in the initial screening of chigger pools for detection of Orientia sp. [74 (link)]. Positive samples were subjected to a nested PCR assay for amplification of the htrA gene [6 (link)]. The PCR amplicons were purified and submitted to Eurofins Genomics (https://www.eurofins.com) for Sanger sequencing in both directions. Paired sequences were aligned to generate a corrected consensus and manually quality-trimmed using Bioedit 7.2.5 [75 ] to produce the final sequence for phylogenetic analyses.

According to the manufacturer’s instructions, next-generation sequencing (NGS) libraries were prepared using the QIAseq-targeted RNA Inflammation and Immunity Transcriptome panel kit (Qiagen, Hilden, Germany). Samples with RIN > 8.5 were used in sequencing analysis. Briefly, 400 ng of RNA was treated with DNAse I and reverse transcribed. Next, 20 ng of cDNA was used in the barcode assignment procedure, followed by magnetic bead purification. Barcoded cDNA was amplified during 8-cycle PCR and purified using magnetic beads. Next, 1st-stage barcoded PCR products were uniquely indexed during the 22-cycle 2nd-stage PCR step. Final PCR amplicons were purified using magnetic beads. NGS libraries were quantified using a Qubit™ High Sensitivity dsDNA Quantification Assay kit (Invitrogen™, Thermo Fisher Scientific, Bleiswijk, The Netherlands) on a Qubit 4.0 fluorometer (Invitrogen™, Thermo Fisher Scientific, Bleiswijk, The Netherlands). Size ranges of NGS libraries were determined using a High Sensitivity DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA) on the Agilent 2100 device. Libraries were appropriately denatured and diluted according to the NextSeq library denaturation and dilution guide. A sequencing run was performed on the Illumina NextSeq 550 sequencer (Illumina, San Diego, CA, USA) using 150-cycle Illumina High Output Kit v2.5 (Illumina, San Diego, CA, USA).