Miseq reagent kit v3 150 cycle

Four Pst-infected wheat samples (17.0504, 17.0505, 17.0506 and 17.0507) were utilised for amplicon sequencing using the MiSeq platform (Illumina, CA, USA). Following DNA extraction and PCR amplification of the 242 selected Pst genes, an equal mass of purified PCR products from each of the four primer pools was combined prior to library preparation, giving a total of 1 μg DNA (250 ng per primer pool; Additional file 1: Table S14). Samples were prepared for sequencing using a KAPA HyperPlus Library Preparation Kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. PCR products were fragmented enzymatically into sizes of approximately 600 bp using a reaction time of 10 min. Each sample was tagged with a unique barcode to enable sample identification. The resulting libraries had insert sizes of 790–911 bp and were made into an equimolar pool of 40 μl prior to sequencing (Additional file 1: Table S14). Libraries were sequenced using an Illumina MiSeq platform and MiSeq Reagent Kit v3 150 cycles (Illumina, CA, USA) following the manufacturer’s instructions.

The libraries were quantified using the KAPA Library Quantification Kit for Illumina (Roche). For one MiSeq run, 24–40 libraries were pooled in equimolar concentration. A final concentration of 18–20 pM library spiked with 5% PhiX (Illumina) was sequenced (75 bp, single read) using a MiSeq Reagent Kit V3—150 Cycles (Illumina). Raw sequencing data is freely accessible under the project identifier SRP279094 from the Sequence Read Archive.

Sequencing of 151 nucleotide-longsingle-end reads was performed on an Illumina MiSeq machine using MiSeq Reagent Kit v3 150 cycles (Illumina). Fastq files were processed and the data were analyzed using custom-made scripts (for details, see Additional file 2, Additional file 3: Table S1). Briefly, BC and ROI sequences were extracted from the reads. Next, a set of genuine BCs was defined as described previously [29 (link)]. Namely, at this step mutant versions of BCs (arisen due to PCR and/or NGS errors) that contain up to 2 nucleotide substitutions were identified and associated with the appropriate intact BCs. Only genuine BCs found in at least 2 reads were kept for the downstream analysis. Then, for each genuine BC the coupled ROI sequences were counted. The ROI sequence found in more than one half of the reads carrying the genuine BC (with Frequency > 0.5) was considered as genuine ROI. ROI sequences differing by 1 nucleotide from the genuine ROI were considered as variations arisen due to PCR and/or NGS errors, while all other ROI sequences were considered to be a result of chimeric PCR.

The DNA quantification was performed with the Quant-iT PicoGreen dsDNA Assay Kit (Life Technology, Carlsbad, CA, USA) using 2.5 µL of sample in 200 µL of reaction mixture. Fluorescence was measured with the Multimode reader infinite M1000 PRO (Tecan, Nänikon, Switzerland). The Nextera XT Library Prep Kit was used to prepare the library for sequencing. The sequencing reaction was performed with the MiSeq Reagent Kit v3 600 Cycles (paired end reads, read length reduced to 250 bp + 250 bp to get high quality reads) for mouse brain samples and with the MiSeq Reagent Kit v3 150 Cycles (paired end reads, 75 bp + 75 bp) for MSV and MSV + 5 samples on a MiSeq sequencer (all Illumina). All amplicons were pooled into one sequencing library.

Viral RNA was extracted from respiratory samples using the QIAamp viral RNA minikit (Qiagen, Hilden, Germany) or the MagMAX CORE nucleic acid purification kit according to the manufacturer's instructions. All eight viral RNA segments of IAV were simultaneously amplified using the SuperScript III one-step RT-PCR system with platinum Taq DNA polymerase (Thermo Fisher Scientific) with two slightly modified universal primers (MBTuni-12v2 [5′-ACG CGT GAT CAG CRA AAG CAG G-3′] and MBTuni-13 [5′-ACG CGT GAT CAG TAG AAA CAA GG-3′]) targeting the highly conserved sequence of viral RNA termini, as reported previously (44 (link)). The multisegment RT-PCR (M-RTPCR) products were processed for sequencing libraries using the NEBNext Ultra II DNA library prep kit for Illumina (New England BioLabs, Ipswich, MA). Next-generation sequencing (NGS) was performed using the MiSeq reagent kit v3 (150 cycles) (Illumina, San Diego, CA). Following automated cluster generation in MiSeq, the sequences were processed and genetic sequence reads were obtained as FASTQ files.

Library preparation was performed using an input concentration of 1/5 µL per sample. We synthesized 24 libraries (8 NC, 8 GC, and 8 ADJ samples) using a TruSeq Small RNA Library Preparation kit (Illumina^®, San Diego, CA, USA), according to the manufacturer’s instructions. The RNA library pool was quantified on ABI 7500 equipment (Applied Biosystem, Foster City, CA, USA) by using a KAPA Library Quantification Kit (KAPA Biosystems, Woburn, MA, USA). The libraries were sequenced on the MiSeq Sequencing System (Illumina^®, San Diego, CA, USA) using the MiSeq Reagent Kit v3 150 cycle (Illumina^®, San Diego, CA, USA).

Sediments from the first five cm were used for DNA extraction and sequencing. Prior to DNA extraction, samples were centrifuged at 10,000× g for 1 min to precipitate the sample and discard the sucrose buffer. Genomic DNA was extracted from 0.25 gr of sediment using the PowerSoil DNA Isolation Kit (Mo Bio Laboratories Inc.) according to the manufacturer’s protocol. DNA was quantified via a NanoDrop lite (Thermo Fisher Scientific) spectrometer. Metagenome libraries were generated using the Nextera XT DNA Library Preparation Kit and sequencing was performed with the MiSeq Reagent Kit v3 (150-cycle) in an Illumina MiSeq platform.