Viral RNA was extracted from 180 μl of each saliva sample using the QIAamp 96 Viral RNA Kit with the QIAcube HT (Qiagen) using the following settings with a filter plate: the lysed sample was premixed eight times before subjecting to vacuum for 5 min at 25 kP and vacuum for 3 min at 70 kPa. Following three washes using the same vacuum conditions above, the samples were eluted in 100 μl AVE buffer followed by a final vacuum for 6 min at 60 kPa. Nine microliters of RNA was used for cDNA synthesis and library preparation using the COVIDSeq Test kit (Illumina) and Mosquito HV Genomics Liquid Handler (SPT Labtech Inc.). The size and purity of the library were determined using the 4200 TapeStation System (Agilent) and the Qubit dsDNA HS Assay Kit (Life Technologies) according to the manufacturer's instructions. Constructed libraries were pooled and sequenced using the NovaSeq. 6000 Sequencing System SP Reagent Kit and the NovaSeq Xp 2‐Lane Kit. Illumina's DRAGEN pipeline was used to derive sample consensus sequences, which were filtered based on a minimum of 70% coverage of the genome.
Dragen pipeline
Manufactured by Illumina
Sourced in United States
The DRAGEN pipeline is a hardware-accelerated secondary analysis solution for next-generation sequencing data. It provides rapid and accurate processing of sequencing data, including alignment, sorting, marking of duplicates, and variant calling.
Automatically generated - may contain errors
Lab products found in correlation
Covidseq test kit, by Illumina (2 mentions)
Qiaamp 96 viral rna kit, by Qiagen (1 mentions)
4200 tapestation system, by Agilent Technologies (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qiacube ht, by Qiagen (1 mentions)
Truseq dna pcr free kit, by Illumina (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Dynamic read analysis for genomics, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Dneasy blood kit, by Qiagen (1 mentions)
Agilent sureselect human all exon v5 kit, by Agilent Technologies (1 mentions)
Novaseq 6000 sequencing platform, by Illumina (1 mentions)
9 protocols using dragen pipeline
1
SARS-CoV-2 Viral RNA Extraction and Sequencing
2
Exome Sequencing Utilizing DRAGEN
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Exome sequencing was performed in a Clinical Laboratory Improvements Amendments (CLIA)-certified environment (GeneDx, Gaithersburg, MD, USA). Analysis was performed using the DRAGEN pipeline (Illumina, San Diego, CA, USA). Copy number variants (CNVs) were called using the DRAGEN CNV pipeline and a normalized segmented depth of coverage model, as previously described (Rockowitz et al. 2020 (link)).
3
Whole-Genome Sequencing and Variant Analysis
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800–1000 ng of genomic DNA of each individual was fragmented to an average length of 350 bp. Library preparation was performed using the TruSeq DNA PCR-free kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Whole-genome sequences were obtained as 150 bp paired-end reads on S4 flow cells using the NovaSeq6000 system (Illumina). The intended average sequencing depth was 30X. The DRAGEN pipeline (Illumina, version 3.6.3 or 3.5.7) was used for alignment and joint variant calling was performed with the Glnexus software (version 1.3.2). Individuals with a 20-fold coverage in less than 96% of the protein-coding sequence were removed as well as related individuals to retain only from related pairs. Variant QC was performed using hail (version 0.2.58). European individuals were selected by performing PCA analysis along with the 1000 genomes data. Finally, annotation was performed using Variant Effect Predictor (VEP, version 101). In the present study, only individuals from the BoSCO and CoMRI substudies were included.
4
Rapid Whole Genome Sequencing for Clinical Use
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Clinical rapid whole genome sequencing (rWGS) of the proband was performed at the Rady Children's Institute for Genomic Medicine in San Diego (CA) (Kingsmore et al., 2022 (link)). Sequence via next‐generation sequencing (NGS) technology was generated from genomic DNA. PCR‐free library preparation was performed prior to rWGS. An average genomic coverage of at least 35x, and/or at least 90% of OMIM genes were achieved 100% of coding base coverage of >10x for each proband. Alignment and variant calling were performed with the Illumina DRAGEN pipeline using the official reference build 37.1 (hg19). CNV calling was performed using a combination of CNV callers. Interpretation of CNVs was focused on variants that overlap or have a boundary that lies within 1 kb of an exon in all coding genes.
5
Transcriptome Analysis of 100k Genomes Project
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Blood was collected from a subset of 100,000 Genomes Project probands in PaxGene tubes to preserve RNA at the time of recruitment. RNA was extracted, depleted of globin and ribosomal RNAs, and subjected to sequencing by Illumina using 100 bp paired-end reads, with a mean of 102M mapped reads per individual. Alignment was performed using Illumina’s DRAGEN pipeline. FRASER226 (link) and OUTRIDER25 (link) were used to detect abnormal splicing events and expression differences with samples run in batches of 500, both run via the DROP pipeline39 (link).
6
Exome Sequencing of EDTA Blood Samples
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Blood samples were collected from subjects in EDTA vacutainers and after collection were immediately stored at − 80 °C. Standard DNA preparation was performed using DNeasy Blood kits (Qiagen, MD, USA). Whole-exome sequencing libraries were generated using the Agilent SureSelect Human All Exon Kit V5 (Agilent, CA, USA) and sequenced on a HiSeq 2500 instrument (Illumina, CA, USA) using standard paired-end sequencing protocol. Raw sequencing reads were stored as FASTQ files and then aligned to the human reference genome (GRCh37) using Illumina’s Dynamic Read Analysis for GENomics (DRAGEN) Pipeline. Resultant BAM files were position-sorted and duplicate reads marked. Single-sample gVCF files were generated by the DRAGEN Germline Pipeline, and joint calling of all samples in the study cohort were performed by DRAGEN Joint Genotyping.
7
Exome Sequencing Utilizing DRAGEN
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Exome sequencing was performed in a Clinical Laboratory Improvements Amendments (CLIA)-certified environment (GeneDx, Gaithersburg, MD, USA). Analysis was performed using the DRAGEN pipeline (Illumina, San Diego, CA, USA). Copy number variants (CNVs) were called using the DRAGEN CNV pipeline and a normalized segmented depth of coverage model, as previously described (Rockowitz et al. 2020 (link)).
8
SARS-CoV-2 Genomic Sequencing from Biological Samples
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Viral RNA from viral transport medium, nasopharyngeal swabs, or saliva was extracted for each sample and used in complementary DNA synthesis as described in the Supplementary Methods. Viral DNA library preparation for next-generation sequencing was performed using the COVIDSeq Test kit (Illumina, San Diego, CA) and Mosquito HV Genomics Liquid Handler (SPT Labtech Inc., Boston MA). Constructed libraries were pooled and sequenced using the Illumina NovaSeq 6000 Sequencing platform. Illumina’s DRAGEN pipeline was used to derive sample consensus sequences, which were filtered based on a minimum of 70% coverage of the genome and 20X sequencing depth.
9
Transcriptome Analysis of 100,000 Genomes Project
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Blood was collected from a subset of 100,000 Genomes Project probands in PaxGene tubes to preserve RNA at the time of recruitment. RNA was extracted, depleted of globin and ribosomal RNAs, and subjected to sequencing by Illumina using 100bp paired-end reads, with a mean of 102M mapped reads per individual. Alignment was performed using Illumina’s DRAGEN pipeline. IGV(48 (link)) was used to inspect sequencing reads and generate Sashimi plots to show splicing junctions supported by 5 or more reads in areas of interest.
FRASER2(49 (link)) and OUTRIDER(50 (link)) were used to detect abnormal splicing events and expression differences with 499 samples used as controls.
FRASER2(49 (link)) and OUTRIDER(50 (link)) were used to detect abnormal splicing events and expression differences with 499 samples used as controls.
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