Qiaseq fx dna library udi kit

RNA was extracted from patient’s blood using a Maxwell RSC simply RNA Blood purification kit according to the manufacturer’s instructions (Promega, USA). Library preparation and sequencing was performed as described^{26 (link)}. In short, cDNA was obtained by using reverse transcriptase with random priming. Following cDNA synthesis, primers based on sequences from the ARTICnetwork were used to generate 400 bp amplicons in two different PCR pools. After merging of pools and amplification, libraries were constructed using QIASeq FX DNA Library UDI Kit following the manufacturer’s instructions (Qiagen GmbH, North Rhine-Westphalia, Germany).
Sequencing was done with Illumina NextSeq® 500/550 using 149-bp paired-end reads with 10-bp indices (Illumina, California, USA). Obtained viral sequences were assembled using CLC Genomics Workbench v20.0.3 (Qiagen GmbH, North Rhine-Westphalia, Germany). SARS-CoV-2 isolate Wuhan-Hu-1 served as the reference genome (Accession NC_045512.2). SARS-CoV-2 variants were identified by uploading FASTA-files on freely accessible databases (http://cov-lineages.org/).

Nasopharyngeal and oropharyngeal swab specimens were collected from symptomatic patients to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was extracted from the specimens using a Qiagen viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Next, real-time RT-PCR was performed on the cycle threshold value of the SARS-CoV-2 target gene (ORF 1b and E) [32 (link)]. For whole-genome sequencing, the cDNA was amplified using the QIAseq SARS-CoV-2 Primer Panel and QIAseq FX DNA Library UDI Kit (QIAGEN, Hilden, Germany).
DNA libraries were extracted using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA), and sequencing was performed on the MiSeq instrument using a MiSeq reagent kit V2 (Illumina, San Diego, CA, USA) to obtain an average genome coverage greater than 1000× for all the isolates. The reads were trimmed and mapped to reference genome MN908947.3 using CLC Genomics Workbench version 20.0.3 (CLC Bio, Aarhus, Denmark). Using the reference genome, single-nucleotide variants (SNVs) were called using the BioNumerics version 7.6 SARS-CoV-2 plugin (Applied Maths, Sint-Martens-Latem, Belgium) [33 (link)].

RNA was extracted from patient’s blood using a Maxwell RSC simply RNA Blood purification kit according to the manufacturer’s instructions (Promega, USA). Library preparation and sequencing was performed as described^{28 (link)}. In short, cDNA was obtained by using reverse transcriptase with random priming. Following cDNA synthesis, primers based on sequences from the ARTICnetwork were used to generate 400 bp amplicons in two different PCR pools. After merging of pools and amplification, libraries were constructed using QIASeq FX DNA Library UDI Kit following the manufacturer’s instructions (Qiagen GmbH, North Rhine-Westphalia, Germany).
Sequencing was done with Illumina NextSeq® 500/550 using 149-bp paired-end reads with 10-bp indices (Illumina, California, USA). Obtained viral sequences were assembled using CLC Genomics Workbench v20.0.3 (Qiagen GmbH, North Rhine-Westphalia, Germany). SARS-CoV-2 isolate Wuhan-Hu-1 served as the reference genome (Accession NC_045512.2). SARS-CoV-2 variants were identified by uploading FASTA-files on freely accessible databases (http://cov-lineages.org/).