Hybrid assembly of Nanopore and Illumina sequences were performed using SPAdes (version 3.6.2) (Bankevich et al., 2012 (link)) with –careful and –nanopore flag. Next, extension was conducted to scaffolding the derived assembly following previous protocol (Ashton et al., 2014 (link)). For comparison, Nanopore sequences were individually assembled by Canu (Koren et al., 2017 (link)) with error rate of 0.035, while the post-QC Illumina reads were assembled by CLC bio Genomics Workbench (version 6.0.4, CLC Bio, Denmark) with default parameters. To avoid the interference of fragmented assembly, contigs shorter than 1 kb or with coverage <20 were filtered before annotation.
Clcbio genomics workbench
Manufactured by Qiagen
Sourced in Denmark, Germany
The CLCbio Genomics Workbench is a bioinformatics software platform designed for the analysis and visualization of genomic data. It provides a comprehensive suite of tools for tasks such as sequence alignment, assembly, annotation, and data exploration.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq buffer, by Promega (2 mentions)
Gotaq polymerase, by Promega (2 mentions)
Varseq, by Golden Helix (1 mentions)
Bigdye terminator v1, by Thermo Fisher Scientific (1 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Baseclear, by Qiagen (1 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (1 mentions)
Qiamp powerfecal dna kit, by Qiagen (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Ribozol, by Avantor (1 mentions)
Nextseq high output 75 cycle sequencing kit, by Illumina (1 mentions)
Rneasy column, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
18 protocols using clcbio genomics workbench
1
Hybrid genome assembly using Nanopore and Illumina
2
Targeted Genome Sequencing and Variant Annotation
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FASTQ files were generated for each patient and imported into CLC Bio Genomics Workbench (version 7.5; CLC Bio, Aarhus, Denmark) for read alignment against the human reference genome (build hg19), variant calling, and coverage statistics for targeted regions. Variants were annotated with customized ANNOVAR annotation scripts (33 (link)). Annotation methods performed at the MHI Biobank (30 (link)) and UPenn (18 (link)) have been described in detail previously.
3
Bioinformatic Analysis of Genomic Variants
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Paired FASTQ files are generated for each sample after sequencing and are imported into CLC Bio Genomics Workbench (CLC Bio, Aarhus, Denmark) for bioinformatic processing. Sequencing reads are aligned to the human reference genome (hg19/GRCh37) and undergo local realignment to improve alignment quality. From the assembled reads, variants are called if there are discrepancies between the reference genome and the sample’s sequence. Following this, VCF and BAM files are created for each sample; these files contain information on the genomic position and zygosity of identified variants, as well as the depth of coverage for each sequencing read. A detailed explanation of the bioinformatic and quality assurance processes have already been reported [21 ].
4
Bioinformatic Analysis of Sequencing Data
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DNA sequence data in the form of FASTQ files were imported into CLC Bio Genomics Workbench (version 8.5; CLC Bio, Aarhus, Denmark) for bioinformatic processing. The sequencing data was aligned to the human reference genome (build hg19), depth of coverage was exported as a BAM file, and any identified variants were exported to VCF files for each patient.
5
Bioinformatic Pipeline for Rare Variant Analysis
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We utilized our standard bioinformatic processing and annotation pipeline (52 (link)). Briefly, CLC Bio Genomics Workbench (version 12.0; CLC Bio, Aarhus, Denmark) was used for the alignment of sequencing reads against the human reference genome (build hg19), the calling of variants, and the generation of VCF and BAM files (52 (link)).
Annotation and analysis of rare variants in SORT1 followed our published procedure (52 (link)). Briefly, single-nucleotide variants (SNVs) were annotated using VarSeq® (version 2.1.1; Golden Helix, Inc., Bozeman, MT). Rare variants were defined as having a minor allele frequency of ≤1% or missing in the Genome Aggregation Database (gnomAD;https://gnomad.broadinstitute.org/ ). Missense, nonsense, insertion/deletion, and splicing variants within SORT1 were retained for analysis. In silico prediction algorithms then pinpointed variants with likely large phenotypic impacts, as described (52 (link)). Our genetic assessment also utilizes polygenic scores for common variants that are determinants of the lipid profile as described (53 (link)).
Annotation and analysis of rare variants in SORT1 followed our published procedure (52 (link)). Briefly, single-nucleotide variants (SNVs) were annotated using VarSeq® (version 2.1.1; Golden Helix, Inc., Bozeman, MT). Rare variants were defined as having a minor allele frequency of ≤1% or missing in the Genome Aggregation Database (gnomAD;
6
Targeted Sequencing Data Analysis
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After sequencing, FASTQ files were generated for each patient and imported into CLC Bio Genomics Workbench (version 10; CLC Bio, Aarhus, Denmark) for alignment against the human reference genome (build hg19), variant calling, and the export of VCF and BAM files that contain the genomic location and zygosity of variants, and the depth of coverage for each sequencing read, respectively.
The VCF files from the European subgroup of the 1KG were downloaded and filtered using PLINK (version 1.9) 19 to obtain the variant information contained within the targeted sequencing regions of our LipidSeq panel.
The VCF files from the European subgroup of the 1KG were downloaded and filtered using PLINK (version 1.9) 19 to obtain the variant information contained within the targeted sequencing regions of our LipidSeq panel.
7
Alu Screening in RP1 Exon 4
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In order to screen for the presence of the Alu element in exon 4 of RP1 distinct pair of primers were designed (forward: 5′-AGGCTTGTTTCCTAGGAGAGGT-3′, reverse: 5′-TTCTGCTTCTTTTTCACTTAGGC-3′) using the CLCbio Genomics Workbench (Qiagen, Hilden, Germany).
PCR amplification was performed in a 20 μl total volume containing 20 ng genomic DNA, 1× GoTaq buffer, 0.5 mM dNTPs, 10 μM of each primer, and 2 units (5 U/μl) of GoTaq polymerase (Promega, Madison, Wisconsin). PCR products were separated following agarose gel electrophoresis. PCR products displaying abnormal size profiles were purified (ExoSAP-IT, USB, Cleveland Ohio) and a sequencing reaction was performed in a total volume of 5 μl using 1 μl primer 3.3 µM, 0.5 μl BigDye Terminator v1.1, and 1 μl of the provided Buffer (Applied Biosystems, Foster City, California) Big Dye terminator cycle sequencing kit on an ABI 3130xl Genetic Analyzer (Applied Biosystems).
For this screening, we used 524 controls from the Tohoku University School of Medicine (N = 95) and the Yokohama City University Graduate School of Medicine (N = 429).
PCR amplification was performed in a 20 μl total volume containing 20 ng genomic DNA, 1× GoTaq buffer, 0.5 mM dNTPs, 10 μM of each primer, and 2 units (5 U/μl) of GoTaq polymerase (Promega, Madison, Wisconsin). PCR products were separated following agarose gel electrophoresis. PCR products displaying abnormal size profiles were purified (ExoSAP-IT, USB, Cleveland Ohio) and a sequencing reaction was performed in a total volume of 5 μl using 1 μl primer 3.3 µM, 0.5 μl BigDye Terminator v1.1, and 1 μl of the provided Buffer (Applied Biosystems, Foster City, California) Big Dye terminator cycle sequencing kit on an ABI 3130xl Genetic Analyzer (Applied Biosystems).
For this screening, we used 524 controls from the Tohoku University School of Medicine (N = 95) and the Yokohama City University Graduate School of Medicine (N = 429).
8
Transcriptomic Profiling of European Populations
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Lappalainen et al.72 (link) report mRNA sequencing of 462 lymphoblastoid cell lines of the 1,000 Genomes Project. RNA sequencing data was downloaded from EBI (http://www.ebi.ac.uk/Tools/geuvadis-das/ ). Analysis was limited to 373 samples of European ancestry. We used CLCBio genomics workbench (Qiagen Inc.) to align reads, calculate RKPM (Reads Per Kilobase of transcript per Million mapped reads) values and analyse the data.
9
16S rRNA Sequencing of Gut Microbiome
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16S sequencing of the V3‐V4 region was performed by BaseClear (Leiden, The Netherlands) and the results were analyzed using the CLC bio genomics workbench (Qiagen, The Netherlands), Microbial Genomics Toolbox. The SILVA 16S v128 99% database was used as reference database. Results show the relative abundances at phylum level for the three different donors.
10
BAC DNA Isolation and Sequencing
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DNA from the selected BACs 783N21 and 650G13 (Yu et al. 2000 ) was isolated as previously described. BAC DNA was pooled and sequenced at the Washington State University Molecular Biology and Genomics Core using Ion Torrent PGM technology. Sequencing reads were de novo assembled using a CLC Bio Genomics Workbench (Qiagen) requiring 95% sequence homology and minimum contig size of 1 kb for assembly.
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