Quick 16s ngs library prep kit

The gDNA samples were processed and sequenced using the ZymoBIOMICS targeted sequencing service at ZymoResearch (Irvine CA). The targeted sequencing of the bacterial 16S rRNA gene was carried out as described [15 (link)], using the Quick-16S NGS Library Prep Kit (ZymoResearch) with custom-designed primers to amplify the V3-V4 region of the 16S rRNA gene, real-time PCR and qPCR fluorescence readings, the Select-a-Size DNA Clean & Concentrator (ZymoResearch), Tapestation (Agilent Technologies) and Qubit (ThermoFisher Scientific). The positive control sample used for library preparation was the ZymoBIOMICS Microbial Community DNA Standard (ZymoResearch). The negative controls included the foSW sample as a control for the DNA extraction method, and a blank for library preparation that was provided by ZymoResearch. The completed library was sequenced using a V3 reagent kit (600 cycles) on Illumina MiSeq, which was calibrated by a 10% spike-in of PhiX DNA. The raw sequence reads were uploaded to the Sequence Read Archive database at NCBI under the BioProject ID PRJNA937707.

For short-read sequencing, 10 ng of genomic DNA samples extracted from a fecal sample were subjected to library construction using the Nextera XT Library Prep Kit (Illumina, CA, USA) or Quick-16S NGS Library Prep Kit (Zymo Research) according the manufacturer’s protocol. Clonal amplification was conducted using the Illumina Miseq platform with the Miseq Reagent kit v3 for 600 cycles. The length of sequenced reads was 2 × 300 nucleotides (nt), and the total read number of individual DNA samples was 50,000–100,000 on average. For long-read sequencing, 10 ng of total genomic DNA were subjected to library construction using the 16S Bar-coding kit (SQK-16S024; ONT, Oxford, UK) according to the manufacturer’s protocol. The bar-coded libraries were loaded and sequenced on MinION flow cells (FLO-MIN106D R9.4.1 using the MinION instrument (ONT). After 17 h, the total read number of individual samples was 60,000–100,000, and the length of sequenced read was 1532 nt in this study.

Briefly, all DNA was quantified using the Qubit^® ds-DNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, Massachusetts, United States). Amplification of the 16S rRNA gene was performed with a modified protocol of the Quick-16S^™ NGS Library Prep Kit (Zymo Research Corp., Irvine, California, United States) with primers for the V3–V4 region of the 16S rRNA gene (covering both Bacteria and Archaea). Additionally, we took advantage of the fluorescence signal to perform a semi-quantitative assessment of the 16S rRNA gene copy number. qPCR provided insight into the relative quantities of 16S rRNA genes, and thus improved the robustness of the DNA sequencing interpretation (more detail available in Supplementary Text S2.2). The 16S rRNA gene-library was sequenced on an Illumina MiSeq platform (Illumina Inc., San Diego, California, United States) at the Lausanne Genomics Technologies Facility (University of Lausanne, Switzerland), applying 10 pM of the 16S rRNA library with a 15% Phi-X spike in a paired-end 300 bp mode. t-tests were achieved with Excel Analysis ToolPak, testing the equality of variance with an F-test beforehand, to assess statistical significance in spatial distribution of microbial biomass (Supplementary Figure S8), and to compare the growth in-between reactors (Supplementary Figure S9). More detail is available in the Supplementary material.

The identification of CLB-associated bacteria was done by 16S rRNA gene sequencing at the V3-V4 hypervariable region by using Next Generation Sequencing (NGS). Sample quality control was done with Qubit dsDNA BR or HS (ThermoFisher). Separate amplicon libraries were prepared for each of the 100 samples by using Quick-16S NGS Library Prep Kit (Zymo Research) according to the manufacturer’s protocol. The sequencing was done with 20 ng of DNA per sample (MiSeq Illumina) with read length 2 × 250 bp, output 25 K clusters per sample. Amplicon libraries targeting the V3–V4 hypervariable regions of the 16S rRNA gene were generated (CeGat GmbH, Germany). The positive and negative controls of the sequencing process were also done. After library preparation, both controls were checked and there were not any abnormalities: negative control was negative (no measurable DNA) and positive control was positive (expected DNA amount and expected fragment length).

Bacterial 16S rRNA gene-targeted sequencing was performed using the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA, United States). Bacterial 16S primers amplified the V1-V2 or V3-V4 region of the 16S rRNA gene. These primers have been custom designed by Zymo Research to provide the best coverage of the 16S gene while maintaining high sensitivity. The sequencing library was prepared using a library preparation process in which PCR was performed in real-time PCR instruments to control cycles and prevent/limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA, United States) and then quantified with TapeStation^® and Qubit^®. The final library was sequenced on Illumina^® MiSeq™ with a v3 reagent kit (600 cycles). The sequencing was performed with > 10% PhiX spike-in.

DNA was extracted from whole O. cornifrons larvae (n = 3 per treatment condition, mancozeb-treated and untreated pollen provisions), we conducted a microbial diversity analysis on these samples, particularly because the highest mortality rates were observed in larvae fed on mancozeb-treated pollen provisions.. Using the DNAZymoBIOMICS^®-96 MagBead DNA Kit (Zymo Research, Irvine, CA), DNA was amplified, purified, and sequenced on Illumina^® MiSeq™ with a v3 reagent kit (600 cycles). Bacterial 16S ribosomal RNA gene-targeted sequencing was performed using the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA), employing primers that target the V3-V4 region of the 16S rRNA gene. Additionally, 18S sequencing was conducted with 10% PhiX spike-in, and amplification was performed using the primer pair 18S001 and NS4.

The 16S rRNA gene sequencing libraries were constructed using the Quick-16S NGS Library Prep Kit (Zymo Research Europe GmbH, Freiburg, Germany) with its included optimized V1-V2 primer pair in runs that included 94 samples, the the positive control, and a negative control. Modified V1-V2 primer pairs were chosen for the analysis due to their advantages in genus-level resolution, length, and ability to detect certain potentially pathogenic genera [23 (link),24 (link),25 (link)]. After pooling, the DNA was quantified with the QuantiFluor dsDNA System on the Quantus Fluorometer (Promega GmbH, Walldorf, Germany) and diluted strictly according to the Illumina protocol for MiSeq sample preparation. For the final library, a loading concentration of 10 pM was chosen and a 10% Illumina v3 PhiX spike-in control was added before running it on the Illumina MiSeq platform with the 500 cycle v2 Illumina MiSeq Reagent Kit. All reagents and equipment for sequencing samples were obtained from Illumina, San Diego, CA, USA. Raw reads have been uploaded to the European Nucleotide Archive; accession PRJEB60950, ERA21159872.