Sequencing data was analyzed using DRAGEN COVID Lineage (version 3.5.9; Illumina Inc., USA) and method as described previously [11 (link),12 (link)]. In brief, sequencing reads of human origin were removed using NCBI Human Read Scrubber algorithm. ARTIC primer sequences were removed from the reads, followed by aligning to the reference genome Wuhan-Hu-1 (GenBank accession number MN908947.3) using DRAGEN (Illumina Inc., USA). Samples with less than 90 amplicons detected are filtered. Variant calling and consensus genome assembly with respect to the reference genome were performed using DRAGEN (Illumina Inc., USA) using default parameters. Nucleotide and amino acid positions were numbered according to the reference genome. All genome sequences analyzed in this study were submitted to GISAID under accession numbers EPI_ISI_13822514 to 13822565 and EPI_ISI_14336314 to 14336324. Pangolin lineages of the consensus genomes were assigned using Pangolin COVID-19 Lineage Assigner (version 4.0.2) [13 (link)]. Phylogenetic clades of the consensus genomes were mapped using Nextclade (version 1.11.0) [14 (link)].
Dragen
Manufactured by Illumina
Sourced in United States
DRAGEN is a high-performance genomic analysis platform that provides rapid and accurate data analysis for next-generation sequencing (NGS) applications. The core function of DRAGEN is to enable efficient processing and analysis of large genomic datasets.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (4 mentions)
Bio it platform, by Illumina (2 mentions)
Truseq nano dna library prep kit, by Illumina (1 mentions)
Hiseq x ten, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Truseq nano dna library prep, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Dragen covid lineage, by Illumina (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Sureselect enzymatic fragmentation kit, by Agilent Technologies (1 mentions)
Maxwell rsc blood dna kit, by Promega (1 mentions)
Unique dual indexes, by Illumina (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
Pronex ngs library quant kit, by Promega (1 mentions)
Rna prep with enrichment l tagmentation kit, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
13 protocols using dragen
1
SARS-CoV-2 Lineage Identification Protocol
2
Whole Genome Sequencing and Variant Calling
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DNA samples were prepared using the TruSeq DNA Nano Library Prep Kit from Illumina. The libraries were pooled and sequenced with the 150-bp paired-end protocol on an Illumina platform to yield an average coverage depth of 30× for the nuclear genome. Raw read alignment to reference genome GRCH38 and variant calling, including single nucleotide substitutions (SNVs), small insertions/deletions (Indels), and structural variants (SVs) with default parameters, were performed using DRAGEN (version 3.10.4, Illumina). SNV and indel annotation were performed by Varvis (Limbus Medical Technologies GmbH; https://www.limbus-medtec.com/ ). Structural variants were annotated with ANNOTSV3.1 and the in-house structural variant database to obtain occurrence frequencies. Genetic variants are described after the Human Genome Variation Society recommendations (https://varnomen.hgvs.org/ ).
3
Whole Genome Sequencing with Illumina NovaSeq
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DNA was prepared for sequencing using the TruSeq DNA PCR-free paired-end protocol (Illumina, San Diego, CA, USA) and WGS was performed on a NovaSeq 6000 instrument (Illumina) with 150 bp paired end sequencing to approximately 30X coverage with v1.5 reagents. Processing of WGS sequencing data was performed using RTA v3.4.4 and bcl2fastq v2.20.0.422, with variant calling (including both small and structural variants) on Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT platform, Ensembl Variant Effect Predictor (VEP) and AnnotSV [25 (link)]. BAM files were visualized using the Integrative Genomics Viewer (IGV) [26 (link)].
4
Whole Genome Sequencing of Affected German Shepherd Dogs
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Three affected GSDs were selected for WGS: a black/tan female with German ancestry (SRR15446412), a white female from the Netherlands (SRR15446416), and a black/tan female from an American service dog breeding colony (SRR15446414). Resequencing of genomes from the latter two dogs was performed using an Illumina HiSeq X Ten, generating 2x150 bp paired-end reads. Total reads generated ranged from 861 to 869 million per sample. Paired-end reads were trimmed, aligned to the indexed reference (CanFam3.1), sorted, and indexed to be viewed in IGV [65 (link)] using the Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT platform [66 (link)]. WGS data for the third affected GSD were generated on an Illumina HiSeq 2000, with 2x125 bp paired-end reads. A total of 584 million reads were trimmed, aligned to CanFam3.1 with Bowtie2 [67 (link)], and sorted and indexed using SAMtools [68 (link)].
5
Whole-Genome Sequencing of PCAT2-Modified Cells
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DNA was isolated from SW480PCAT2-KI colon cancer cells using phenol:chloroform:isoamyl alcohol method. The quality of the DNA was tested using 2100 Bioanalyzer (Agilent, USA). DNA was sequenced on NovaSeq 6000 Standard SP run using TruSeq Nano DNA Library Prep (Illumina, USA) and paired-end sequencing mode. The sample was mapped, and variants were called using DRAGEN (Illumina, USA). The insert sequence was identified from the whole-genome sequencing reads using a blast search algorithm against a custom reference with the insert plasmid sequence.
6
Whole Genome Sequencing Annotation and Analysis
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Whole genome sequencing (WGS) was done at 30X coverage for the 1653 individuals from the Gabriella Miller Kids First project, by the genomics platform of the Broad Institute. The variant call format (VCF) files of WGS were generated using the Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform (Illumina, San Diego, CA), aligned to the GRCh38/hg38 human genome assembly. For the validation dataset, WGS VCF files of participating individuals were extracted from the TOPMED database directly. The annotations for the variants were generated using the ANNOVAR software developed by our group [5 (link)], and the variants were further divided into three groups based on their genomic locations, i.e., variants in coding regions, variants in non-coding variants including intronic variants, variants in untranslated regions (UTR) or in non-coding RNAs, and variants in intergenic regions.
7
Whole Genome Sequencing and Variant Calling
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Analysis DNA was isolated, and 2x100 or 2x150 nucleotide rWGS was performed to ~45-fold coverage as previously described (4 (link)). Sequence alignment to the reference human genome and nucleotide variant calling was by DRAGEN (Illumina Inc, San Diego, CA) (4 (link)). An automated copy number variation pipeline was implemented in July 2017, which identified CNVs with a combination of the tools Manta and CNVnator (56 (link), 57 (link)). Prior to July 2017, any CNV diagnoses were made via manual inspection of raw genomic data. Variants were annotated, analyzed, and interpreted with Opal Clinical (Fabric Genomics, Oakland, CA). All causative SNVs were confirmed by Sanger sequencing and CNVs were confirmed by MLPA and qPCR or aCGH. No variants failed confirmation.
8
Transcriptomic Analysis of L. rohita
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The raw binary base call (BCL) files were converted into FASTQ files using Illumina Dragen (--bcl-conversion-only) and the sequencing files were evaluated using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). The raw sequences data have been submitted to NCBI Short Read Archive (SRA) (Table 1 ). The high-quality reads (i.e., Q-value >30), were used to map against reference genome of L. rohita (Jayanti breed) (GenBank assembly accession: GCA_004120215.1) downloaded from NCBI with Dragmap (https://github.com/Illumina/DRAGMAP ). FeatureCounts was used to count the abundance of individual reads that mapped to genomic features.
9
Whole-Genome Sequencing for Translocation Breakpoint Mapping
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In order to clarify previous findings and fine‐map the translocation breakpoints, DNA was extracted and paired‐end whole‐genome sequencing (2 × 150 bp) was performed using a NovaSeq 6000 (Illumina) platform. Library preparation for sequencing was performed using the TruSeq PCR‐free (Illumina) kit. The mean genome coverage achieved by NGS was 34.3X. Bioinformatics data analysis as well as a structural variant calling was performed using DRAGEN (Illumina) software. Integrative Genomics Viewer (IGV) software was used for data visualization. Breakpoint detection and fine‐mapping was performed by the manual exploration of the region of interest on IGV (Figure 3 ).
10
Whole Exome Sequencing of Blood Samples
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Genomic DNA was extracted from peripheral blood mononuclear cells using the Maxwell® 48 Instrument (Promega,) and the Maxwell® RSC Blood DNA Kit (AS1400). DNA quantification was performed on Qubit® 3.0 Fluorometer (Broad range Kit, Invitrogen, Q32853), the Nanophotometer® P-Class 300 instruments.
DNA was fragmented using SureSelect Enzymatic Fragmentation kit (Agilent). NGS library were prepared using SureSelect XT HS/Low Input Kit with All Exome V7 RNA Oligos (Agilent). Both DNA fragmentation and library preparation were automated on Hamilton MicroLab STAR M technology.
Library concentration and quality were assessed by Qubit® 3.0 Fluorometer and the 2100 Bioanalyzer Instruments, respectively.
WES was performed on Illumina NovaSeq 6000, S2 flow cells, with a mean coverage 114x.
Reads were aligned against GRCh37 reference. Variant calling was executed with germline pipeline of Dynamic Read Analysis for GENomics (DRAGEN, Illumina). Single nucleotide variants (SNVs) were annotated using ANNOVAR 3.1.2 [20 (link)].
DNA was fragmented using SureSelect Enzymatic Fragmentation kit (Agilent). NGS library were prepared using SureSelect XT HS/Low Input Kit with All Exome V7 RNA Oligos (Agilent). Both DNA fragmentation and library preparation were automated on Hamilton MicroLab STAR M technology.
Library concentration and quality were assessed by Qubit® 3.0 Fluorometer and the 2100 Bioanalyzer Instruments, respectively.
WES was performed on Illumina NovaSeq 6000, S2 flow cells, with a mean coverage 114x.
Reads were aligned against GRCh37 reference. Variant calling was executed with germline pipeline of Dynamic Read Analysis for GENomics (DRAGEN, Illumina). Single nucleotide variants (SNVs) were annotated using ANNOVAR 3.1.2 [20 (link)].
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