Copy number Variation Sequencing (CNVseq) was performed as previously described (Gao et al., 2019 (link)). Briefly, the genomic DNA was fragmented by sonication (Covaris, United States) into 200–300 bp fragments and checked using agarose gel electrophoresis. After genomic library preparation, DNA samples were subsequently sequenced on an Illumina NovaSeq 6000 series sequencer (Illumina, San Diego, CA, United States). Raw image files were processed using BclToFastq (Illumina) for the base calling and raw data generation. The reads were then mapped to the GRCh37/hg19 human reference genome using the BWA software (Li and Durbin, 2010 (link)). Variant calling for CNVs ≥100 kb was performed using an in-house pipeline, and the candidate CNVs were filtered and detected using public CNV databases (Decipher, ClinVar, OMIM, DGV, and ClinGen). The pathogenicity of CNVs was classified according to the American College of Medical Genetics and Genomics guidelines (Riggs et al., 2020 (link)).
Novaseq 6000 series sequencer
Manufactured by Illumina
Sourced in United States
The NovaSeq 6000 series is a high-throughput DNA sequencing system designed for genomic research. It is capable of generating high-quality sequencing data with a range of read lengths and throughput levels to support various applications.
Automatically generated - may contain errors
Lab products found in correlation
Bcltofastq, by Illumina (2 mentions)
Xgen exome research panel v1, by Integrated DNA Technologies (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sonication, by Covaris (1 mentions)
Dna blood mini kit, by Qiagen (1 mentions)
Genomic dna extraction kit, by Qiagen (1 mentions)
Dna extraction kit, by Tiangen Biotech (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
10 protocols using novaseq 6000 series sequencer
1
Comprehensive Copy Number Variation Analysis
2
Whole Exome Sequencing for Genetic Variants
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Genomic DNA of peripheral blood was extracted using a DNA Blood Mini kit (Qiagen) according to the kit instructions. A library of whole exomes was built using Roche Nimble Gen Seq EZ Exome Enrichment Kit V2.0 and Seq EZ Exome Enrichment Kit V2.0 capture probes (Roche). High-throughput sequencing was performed by 150-bp paired-end sequencing with Illumina NovaSeq 6000 series sequencer (PE150). The clean data were aligned to the NCBI human reference genome (hg19) using Burrows-Wheeler Aligner-Maximal Exact Match (BWA-MEM) algorithm.48 (link) Variants were called using HaplotypeCaller (https://gatk.broadinstitute.org/ )49 (link) and annotated with databases such as Online Mendelian Inheritance in Man (OMIM), Exome Aggregation Consortium (ExAC), and predicted by online analysis programs. Mutations of XPNPEP3 (GenBank: NC_000022.11 ) identified in probands and patients were validated by Sanger sequencing. Primers were listed in Table S2 .
3
Whole Exome Sequencing Variant Filtration
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Using IDT The xGen Exome Research Panel v1.0 full exon capture chip and Illumina NovaSeq 6000 series sequencer, the whole exome of the proband was sequenced with high throughput, and the target sequence coverage was not less than 99%. Variants were removed if the following conditions were met: (a) the minor allele frequency is greater than or equal to 1% in ExAC Browser, gnomAD, or the 1,000 genome Project; (b) the variant is not predicted deleterious by SIFT, PolyPhen‐2, and MutationTaster tools; (c) the variant in noncoding exons, 3′ or 5′ untranslated regions, or intronic sequences except canonical splice sites.
4
Whole-Exome Sequencing with Roche Enrichment
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The whole-exome library was constructed using the Roche Nimble Gen Seq EZ Exome Enrichment Kit with capturing probes V2.0 (Roche, USA), and the DNA of total exons and their flanking introns were enriched. High-throughput sequencing was performed on an Illumina NovaSeq 6000 series sequencer (Illumina, USA), with at least 99% of target sequences sequenced at a 150 × reading depth.
5
Whole-Genome Sequencing for Syndromic Disorders
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Whole-genome low-coverage sequencing was performed to identify CNVs in patients with syndromic short stature and intellectual disability. Genomic DNA was extracted from peripheral blood samples and sheared to 200–300 bp fragments by sonication, followed by electrophoresis analysis for quality control. The fragments were then end-repaired and A-tailed in preparation for ligation to adapters. The ligates were amplified by ligation-mediated PCR for 4–6 rounds. High-throughput sequencing was performed on an Illumina NovaSeq 6000 series sequencer (Illumina, San Diego, CA, USA). Raw image files were processed by BclToFastq (Illumina) for base calling and raw data generation. Reads were mapped to reference genome hg19 using BWA software. CNVs of 100 kb or more in length were detected using Chigene independently developed software packages for CNV detection.
CNV intervals were detected and screened according to public CNV databases and local databases. Decipher, ClinVar, ClinGen, and the Database of Genomic Variants (DGV) were used as references to annotate the pathogenic classification of each screened CNV. The biological harm and related phenotypes of CNVs were assessed by annotated information and frequency databases according to the ACMG practice guidelines and 2011 and 2013 CNV diagnostic guidelines [14 (link), 15 (link)].
CNV intervals were detected and screened according to public CNV databases and local databases. Decipher, ClinVar, ClinGen, and the Database of Genomic Variants (DGV) were used as references to annotate the pathogenic classification of each screened CNV. The biological harm and related phenotypes of CNVs were assessed by annotated information and frequency databases according to the ACMG practice guidelines and 2011 and 2013 CNV diagnostic guidelines [14 (link), 15 (link)].
6
Exome Sequencing for Novel Mutation Detection
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Peripheral blood samples were collected from the proband and her parents in EDTA-treated tubes. The genomic DNA was extracted using the Blood Genome Column Medium Extraction kit (CWBio), according to the manufacturer's instructions. The extracted DNA was then subjected to the quality control process using a Qubit 2.0 fluorimeter and 0.8% agarose gel electrophoresis. Subsequently, protein-coding exome enrichment was performed using the xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc.). This panel consists of 429,826 individually synthesized and quality-controlled probes that target 39 Mb protein-coding regions (19,396 genes) of the human genome and cover 51 Mb of end-to-end tiled probe space. Furthermore, high-throughput sequencing was carried out on a NovaSeq 6000 series sequencer (PE150; Illumina, Inc.), and >99% of the target sequences were sequenced. The sequencing process was performed by Chigene Translational Medicine Research Center Co., Ltd. A search was conducted in the HGMDpro database for the novel mutation (https://digitalinsights.qiagen.com/products-overview/clinical-insights-portfolio/human-gene-mutation-database/ ).
7
Whole Exome Sequencing Protocol
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A total of 2 mL fasting blood was collected from the proband and her parents between 08:00 and 10:00 hours into ethylenediaminetetraacetic acid-treated tubes. Genomic DNA was extracted using the Blood Genome Column Medium Extraction Kit (Kangweishiji, Beijing, China) according to the manufacturer’s instructions. For whole exome library construction, protein-coding exome enrichment was performed using xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc., Coralville, IA, USA). This consists of 429,826 individually synthesized and quality-controlled probes, targeting a 39-Mb protein-coding region (19,396 genes) of the human genome and covering 51 Mb of end-to-end tiled probe space. High-throughput sequencing was performed by an Illumina NovaSeq 6000 series sequencer (PE150; Illumina, San Diego, CA, USA), and not less than 99% of the target sequence was sequenced.
8
Fetal Umbilical Cord Blood Cytogenomic Analysis
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Fetal umbilical cord blood samples were obtained for chromosome G band karyotype analysis according to standard operating procedures. Chromosomal description and naming were based on the International System for Human Cytogenetic Nomenclature (Simons et al., 2013 ).
Genomic DNA (gDNA) was extracted according to the instructions of genomic DNA extraction kit (Qiagen Inc., Hilden, Germany) from the fetal umbilical cord blood. Library preparation was in accordance with standard operating procedures. High‐throughput sequencing was performed on Illumina NovaSeq 6000 series sequencer (Illumina Inc., San Diego, USA). Reads were mapped to reference genome hg19 using the BWA software. Chromosomal DNA affected at >100 kb was identified as a copy number variation (CNV). CNVs were classified according to American College of Medical Genetics (AGMG) guidelines (Kearney et al.,2011 ).
Genomic DNA (gDNA) was extracted according to the instructions of genomic DNA extraction kit (Qiagen Inc., Hilden, Germany) from the fetal umbilical cord blood. Library preparation was in accordance with standard operating procedures. High‐throughput sequencing was performed on Illumina NovaSeq 6000 series sequencer (Illumina Inc., San Diego, USA). Reads were mapped to reference genome hg19 using the BWA software. Chromosomal DNA affected at >100 kb was identified as a copy number variation (CNV). CNVs were classified according to American College of Medical Genetics (AGMG) guidelines (Kearney et al.,
9
Trio Whole-Exome Sequencing Protocol
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For trio whole‐exome sequencing (WES), genomic DNA was extracted from the proband and their parents using a DNA extraction kit (Tiangen, Beijing, China), and the purity and concentration of DNA samples were determined to 50–250 ng/μL using a nucleic acid quantification instrument (Nanodrop2000) and stored at −20°C. The xGen Exome Research Panel v2.0 (IDT, USA) was used for exome library constructing, followed by next‐generation sequencing on the NovaSeq 6000 series sequencer (Illumina, USA). Data cleaning and quality control were performed following the manufacturer's instructions. Burrows‐Wheeler Aligner was used for aligning the reads to the Ensemble GRCh37/hg19 reference genome. Base quality score recalibration together with SNP and short indel calling was conducted using the GATK software package. PolyPhen2 and MutationTaster software was applied for protein function prediction and the pathogenicity of genetic variants was annotated according to the American College of Medical Genetics and Genomics (ACMG) clinical practice guidelines (Richards et al., 2015 (link)). The predicted structure of IARS1 (PDB: AF‐P41252‐F1) was downloaded from AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/ ) (Jumper et al., 2021 (link)). The constructed model structure was visualized and analyzed using PyMOL software(www.pymol.org ).
10
Targeted NGS for Inherited Cardiomyopathy
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We collected the EDTA-treated peripheral blood from the consanguineous Chinese family members with informed consents. The peripheral blood genomic DNA was extracted using the DNeasy kit (Qiagen). The concentration and quality of extracted DNA samples were measured by Nanodrop 2000 spectrophotometer (Thermo scientific). Targeted NGS, including DNA library construction, capture, and sequencing, was performed by MyGenostics Gene Technologies (Beijing, China). Custom targeted gene enrichment was performed using the GenCap Custom Enrichment Kit (MyGenostics) according to the manufacturer's protocol. High-throughput sequencing was performed by Illumina NovaSeq 6000 series sequencer (PE150), and not less than 99% of target sequence was sequenced. We excluded single nucleotide polymorphisms of a quality score <20, a strand bias >60, in the exomes generated from 535 unaffected control samples and minor allele frequency (MAF) >0.2%, 0.4%, 0.05%, respectively for HCM, DCM, and ARVC (1,000 genomes, dbSNP, ESP, ExAC, and Chigene in-house MAFs database).
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