Dragen covid lineage app

Samples with Ct values below 32 were sequenced using the ARTIC protocol and the Illumina DNA Prep library kit on a MiSeq instrument (https://www.protocols.io/view/sars-cov-2-sequencing-on-illumina-miseq-using-arti-bssjnecn). Data generated using the Integrated DNA Technologies ARCTIC V4 primer panel were analyzed using the Illumina® DRAGEN COVID Lineage App, which uses a customized version of the DRAGEN DNA pipeline to perform Kmer-based detection of SARS-CoV-2. The app aligns reads to a reference genome, calls variants, and generates a consensus genome sequence. Lineage/clade assignments were also confirmed using NextClade (https://clades.nextstrain.org/, version 1.14.0) and Pangolin COVID-19 Lineage Assigner (https://pangolin.cog-uk.io/, version 3.1.20) by uploading obtained FASTA files^{76 (link),77 (link)}. Consensus sequences generated and related metadata for environmental samples were shared publicly on Global Initiative on Sharing All Influenza Data (GISAID) (www.gisaid.org) (EPI_ISL_8879388 and EPI_ISL_8879389), the principal repository for SARS-CoV-2 genetic information.

Two different bioinformatic approaches were used for whole genome sequence analysis: an open source pipeline based on IVAR (GitLab, 2020 ) and the DRAGEN COVID Lineage App available version (Illumina^®). Both pipelines map quality- and primer- trimmed viral reads to the hCoV-19/Wuhan/WIV04/2019 reference sequence genome (MN908947.3/NC_045512.2) and result in the generation of consensus whole genome sequences.
Consensus sequences covering at least 75% of the reference sequence and with a median coverage greater than 100 reads were considered for lineage assignment. For this purpose, the Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) tool was used (Github, 2020 ), employing the latest version and the most updated lineage database available at the time of assignment. Detection of additional spike amino acid substitutions was conducted by using the Basic Local Alignment Search Tool (BLAST) and the spike protein of the hCoV-19/Wuhan/WIV04/2019 strain as reference.

The COVIDSeq commercial platform on an Illumina NextSeq500 machine with the V4 primer pool was used to genotype SARS-CoV-2 positive samples. Briefly, the protocol amplifies 98 viral targets based on the ARTIC V3 protocol (https://artic.network/ncov-2019, accessed on 31 January 2024). The presence of SARS-CoV-2 in the samples was established with a threshold of >90 targets detected per sample using default thresholds. Then, variant typing, both Pangolin and NextClade systems, was conducted using the DRAGEN COVID lineage app, which is freely available at Illumina BaseSpace (https://basespace.illumina.com/) accessed on 31 July 2023.

The raw data in the form of binary base call format (.bcl files) was generated from the NextSeq 550 instrument. These raw files were converted, demultiplexed to fastq file using bcl2fastq (Illumina, v2.20) and were aligned against the SARS-CoV-2 reference genome (NC_045512.2). The alignment of unmapped reads to a reference genome and generation of a consensus genome sequence was done within the custom Illumina BaseSpace Sequence Hub. The lineages nomenclature for each sequence was retrieved using Illumina DRAGEN COVID Lineage App (v3.5.3) following the default parameters. The minimum accepted alignment score was set to 12 and results with scores <12 were discarded. The coverage threshold and virus detection threshold were set to 20 and 5 respectively. The variant calling target coverage which specifies the maximum number of reads with a start position overlapping any given position was set at 50.
Among 747 sequenced, 612 (81.9%) samples with genomic coverage > 80% were finally selected for downstream analysis. The median genomic coverage of quality passed samples was 98.8 while the median sequencing depth was 1805 (Supplementary Figs. S7 to S10 in the supplemental material).

The SARS-CoV-2 positive samples (n = 1942) were sequenced using an Illumina sequencing machine (COVIDSeq™ Test). Illumina® DRAGEN COVID Lineage App (DRAGEN DNA pipeline version 3.5.7) was used to perform K_mer-based detection of SARS-CoV-2. The viral reads obtained were aligned to the Wuhun-Hu-1 reference genome (NC_045512.2) to call nucleotide variants and generate consensus genome sequences. The Pango lineage assignment was performed using Pangolin (https://pangolin.cog-uk.io/) and NextClade (https://clades.nextstrain.org/).

Two fastQ files were generated for each patient (Read 1, R1; and, Read 2, R2) after the sequencing procedure. All data were uploaded to BaseSpace Sequence Hub (Illumina, USA) to perform the mapping to the SARS-CoV-2 reference genome (Wuhan; NC_045512.2) and to report the genome coverage and sequencing depth using the DRAGEN COVID Lineage App (v3.5.2, Illumina, USA). This App performs Kmer-based detection followed by Map/Align, Variant Calling, and Consensus Sequence generation. Furthermore, it performs lineage/clade determination and mutation characterization by using updated Pangolin (https://cov-lineages.org/pangolin.html) and NextClade (https://clades.nextstrain.org/) nomenclatures. Only those sequences with good quality (>80% genome coverage and minimum depth of 100X) [12 ] were used for molecular characterization of the S protein in comparison with the reference genome (NC_045512.2), using MEGA v6 [13 (link)], and also uploaded to GISAID database [14 (link)]. Additionally, the evolutionary divergence within and between genetic groups of whole-genome and Spike sequences depending on the pandemic wave was performed with p-distance method in MEGA v6 [13 (link)].