Nextera xt dna library prep protocol

The RNA extracted from samples of vaccinated and nonvaccinated animals with a RT-qPCR Ct value lower than 37 were used for whole genome amplification [12 (link),37 (link)]. The PCR conditions were: 0.2 μM of forward and reverse primers MBTuni-12 and MBTuni-13, respectively, 2.5 μL of extracted RNA and 0.5 μL of SuperScript^® III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Moreover, for the biggest segments (PB2, PB1 and PA), amplification was enhanced by a second amplification with the same conditions but adding a modification of forward primer (MBTuni12G) [38 (link)].
Those samples with the eight segments amplified were selected for sequencing [37 (link)] by Illumina technology. The Nextera-XT DNA Library Prep protocol (Illumina^®, San Diego, CA, USA) was followed for multiplexed sequencing libraries and sequencing was performed by Miseq Reagment Kit v2 in a 150-cycle paired-end run, on a Miseq Instrument (Illumina^®, San Diego, CA, USA).
Sequencing data were deposited at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ (accessed on 10 May 2022)) with the accession number (PRJNA853173).

The A/Swine/Spain/01/2010 (H1N1) inoculum and samples with an RT-qPCR Ct value lower than 35 were used for whole-genome sequencing, by simultaneous amplification of the eight influenza RNA segments [33 (link)]. In addition, to enhance the amplification of the longest segments (PB1, PB2, and PA), a second amplification reaction was performed, according to a previously described protocol [34 (link)]. The amplification reaction conditions were: 0.2 μM of forward primer (MBTuni-12 or MBTuni12G), 0.2 μM of reverse primer (MBTuni-13), 2.5 μL of extracted RNA, and 0.5 μL of SuperScript^® III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA).
Samples with the eight influenza segments amplified [33 (link)] were sequenced by Illumina technology. Multiplexed sequencing libraries were created following the Nextera-XT DNA Library Prep protocol (Illumina^®, San Diego, CA, USA). Libraries were sequenced using a Miseq Reagment Kit v2 in a 150 cycle paired-end run, on a Miseq Instrument (Illumina^®, San Diego, CA, USA). Finally, Illumina adapters were automatically removed from FASTQ files. Sequencing data were deposited at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ (accessed on 14 September 2021)), with the accession number (PRJNA763061).

The complete transcribed spacer region [22 (link)] was amplified using the barcoded primers ITS1 and ITS4 [23 ]. PCR was carried out in 50 μL reaction volumes using 25 μL of Q5^® High-Fidelity 2× Master Mix (NEB, Ipswich, UK), 2.5 µL of each primer (10 µM), 2 μL of template DNA, and 18 µL of nuclease-free water. An initial denaturation step of 2 min at 95 °C was followed by amplification for 35 cycles under the following conditions: 30 s at 95 °C, 30 s at 55 °C, and 60 s at 72 °C. A final 5 min extension at 72 °C completed the protocol. The final PCR products were separated on a 1.3% agarose gel (Serva, Heidelberg, Germany) and purified with a NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The cDNA amplicon library was prepared according to the Illumina Nextera XT DNA Library Prep protocol (Illumina, San Diego, CA, USA). The final library was subjected to a quality check using a Fast qPCR Library Quantification Kit (MCLAB, San Francisco, CA, USA) and sequenced using MiniSeq (Illumina) (2 × 150 base reads) with a MiniSeq Mid Output Kit (300 cycles) (Illumina). Negative controls were included during the extraction, amplification, and sequencing to evaluate potential contamination throughout the entire process.