DNA was extracted from all samples using the QIAamp® UCP Pathogen DNA Kit (Qiagen), following the manufacturer’s instructions. Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma) [23 (link)]. Libraries were constructed for DNA using the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) [24 (link)]. The library quality was assessed using the Qubit dsDNA HS Assay kit followed by a high-sensitivity DNA kit (Agilent) on the Agilent 2100 Bioanalyzer. Library pools were then loaded onto the Illumina NextSeq 550Dx sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library. For negative controls, we prepared swabs from 10 healthy donors and added 105 HeLa cells/mL using the same protocol. Sterile deionized water was extracted with specimens to serve as non-template controls [24 (link), 25 (link)].
Nextseq 550dx sequencer
Manufactured by Illumina
Sourced in United States
The NextSeq 550Dx sequencer is a laboratory instrument designed for high-throughput DNA sequencing. It utilizes sequencing-by-synthesis technology to generate genomic data. The core function of the NextSeq 550Dx is to perform automated DNA sequencing and data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs assay kit, by Agilent Technologies (3 mentions)
Nextera xt dna library prep kit, by Illumina (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
High sensitivity dna kit, by Agilent Technologies (3 mentions)
Qiaamp ucp pathogen dna kit, by Qiagen (2 mentions)
Benzonase, by Qiagen (2 mentions)
Tween 20, by Merck Group (2 mentions)
Nextera xt kit, by Illumina (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Variant interpreter, by Illumina (1 mentions)
Veriseq nipt solution v2, by Illumina (1 mentions)
Ngs technology, by Illumina (1 mentions)
Nextera dna exome, by Illumina (1 mentions)
Qiaamp dna ffpe kit, by Qiagen (1 mentions)
Dna prep m tagmentation kit, by Illumina (1 mentions)
Kapa dna hyperprep kit, by Roche (1 mentions)
Nextseq 550dx platform, by Illumina (1 mentions)
Truseq stranded mrna, by Illumina (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
2100 biological analyzer, by Agilent Technologies (1 mentions)
12 protocols using nextseq 550dx sequencer
1
DNA Extraction and Sequencing for Pathogen Detection
2
Multiomic Pathogen DNA Extraction and Sequencing
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DNA was extracted from all samples using a QIAamp® UCP Pathogen DNA Kit (Qiagen) following the manufacturer’s instructions. Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma) [6 (link)]. 10 nanograms DNA samples were used for library construction through Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) [7 (link)]. Library was qualitatively assessed by Qubit dsDNA HS Assay kit, followed by High Sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq 550Dx sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library. For negative controls, we also prepared peripheral blood mononuclear cell(PBMC) samples with 105 cells/mL from healthy donors in parallel with each batch, using the same protocol, and sterile deionized water was extracted alongside the specimens to serve as non-template controls (NTC) [7 (link), 8 (link)]. DNA-free water went through DNA extraction and mNGS analysis as a blank control group to assess the degree of background contamination associated with DNA extraction kit and sequencing reagents together.
3
Whole Exome Sequencing for Retinal Disorder Diagnosis
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Whole Exome Sequencing (WES) was performed in case I:2 on genomic DNA extracted from peripheral blood. Target DNA regions were enriched by Nextera DNA Exome probes (Illumina, San Diego, CA, USA) and then sequenced on NextSeq550Dx sequencer (Illumina). Sequencing reads were aligned to the human reference genome (UCSC hg19) by BWA (v0.7.7-isis-1.0.2) (Illumina). Variant calling was performed by GATK Variant Caller (v1.6-23-gf0210b3). The DNA variants were annotated by Variant Interpreter (v.2.13.0.20) (Illumina). Variants’ mapping in genes associated to retinal disorders was prioritized and then filtered by MAF < 0.01 (GnomAD v2.1). Filtered variants were classified according to ACMG-AMP criteria [9 (link)]. The filtered variant was tested by Sanger sequencing both in case I:2 as well as in cases II:1, II:2, III:1 and III:2, with the following primers’ pair (annealing temperature: 56 °C, extension time: 30″):
CHM Ex10 FW: 5′-AGCCCTCAAAATAGCAACAAG-3′
CHM Ex10 Rv: 5′-CCCTAAAACCAGACCCTGTA-3′
To analyze the functional effect of the CHM splicing variant, mRNA from peripheral blood of case II:2 was retro-transcribed into cDNA and then sequenced by the Sanger technique with the following primers’ pair, spanning from CHM exon 8 to 13:
CHM Ex8-13 FW: 5′-CAATGACATCAGAGACAGCCA-3′
CHM Ex8-13Rv: 5′-TGTGCAAGTCAAATGAACCAA-3′
CHM Ex10 FW: 5′-AGCCCTCAAAATAGCAACAAG-3′
CHM Ex10 Rv: 5′-CCCTAAAACCAGACCCTGTA-3′
To analyze the functional effect of the CHM splicing variant, mRNA from peripheral blood of case II:2 was retro-transcribed into cDNA and then sequenced by the Sanger technique with the following primers’ pair, spanning from CHM exon 8 to 13:
CHM Ex8-13 FW: 5′-CAATGACATCAGAGACAGCCA-3′
CHM Ex8-13Rv: 5′-TGTGCAAGTCAAATGAACCAA-3′
4
Nextera XT Library Construction and Sequencing
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Libraries were constructed by using the Nextera XT kit (Illumina) following the manufacturer manual. Illumina NextSeq-550Dx sequencer was employed for the sequencing reaction to gain the sample sequence information in the sample. The sequencing depth of each sample ≥ 20 million reads was sequenced using a 75-cycle single-end sequencing strategy.
5
Nextseq 550Dx Single-end Sequencing
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Libraries were constructed for the DNA and cDNA samples using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) (17 (link)). The initial input of DNA is 5–100 ng. Firstly, DNA needs to be fragmented to obtain 150–250 bp inserts, followed by terminal repair and adapter connection, and finally, library amplification to construct a library that meets the requirements of sequencing. Library was quality assessed by Qubit dsDNA HS Assay kit followed by High Sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq 550Dx sequencer for 75 cycles of single-end sequencing to generate ~20 million reads for each library. For negative controls, we also prepared PBMC samples with 105 cells/mL from healthy donors in parallel with each batch, using the same protocol, and sterile deionized water was extracted alongside the specimens to serve as non-template controls (NTC).
6
Rapid mNGS Protocol for Microbial Identification
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The Tiangen Magnetic DNA Kit (Tiangen Biotech, DP316) was used to extract the genomic DNA from all samples following the manufacturer’s manual. The extracted nucleic acid samples were used for the generation of DNA libraries using the Nextera XT kit (Illumina) according to the manufacturer’s operational manual. Libraries were sequenced using the Illumina NextSeq-550Dx sequencer. Sterile nuclease-free deionized water was extracted together with samples to serve as negative control. Commercial suspension of bacteria in the assay kit served as positive control. The sequencing depth of each sample was ≥ 20 million reads. High-quality sequencing data were generated by removing low-quality reads, duplicate reads, and shorter reads (<35 bp). Then the human host reads were subtracted using Burrows-Wheeler Aligner software to map to a human reference genome (hg19). The remaining data were aligned with the microbial genome database. Matching microbial reference genomes were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/
7
Prenatal Aneuploidy Screening via cfDNA
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We only tested blood samples where the collection of POC was adequate for the CMA. Analysis of circulating cfDNA in maternal plasma was undertaken by a whole genome sequencing (WGS) from a single tube of maternal blood. The analysis was performed by VeriSeq NIPT Solution v2 powered by Illumina NGS technology. Test menu options were expanded to include common aneuploidies (chromosomes 21, 18, and 13), all rare autosomal aneuploidies (RAAs), sex chromosome aneuploidies (SCAs), and partial deletions and duplications, referred to as copy number variation (CNVs), ≥7 Mb in size. The VeriSeq NIPT Solution v2 incorporates workflow, instrument, and software innovations performing clinical prenatal aneuploidy screening.
The workflow of the analysis Ided cIDNA isolation from maternal plasma, library preparation, next-generation sequencing (NGS), data analysis, interpretation, and reporting. DNA libraries will be sequenced by NextSeq550DX sequencer (Illumina, San Diego, CA, USA).
The workflow of the analysis Ided cIDNA isolation from maternal plasma, library preparation, next-generation sequencing (NGS), data analysis, interpretation, and reporting. DNA libraries will be sequenced by NextSeq550DX sequencer (Illumina, San Diego, CA, USA).
8
Whole Exome Sequencing for Rare Disease Diagnosis
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Whole exome sequencing (WES) was carried out on genomic DNA extracted from peripheral blood by the Nextera DNA Exome (Illumina, San Diego, CA, USA) on a NextSeq550Dx sequencer (Illumina). Sequencing reads were aligned to the human reference genome (UCSC hg19) by BWA (v0.7.7-isis-1.0.2) (Illumina). Variant calling was performed by GATK Variant Caller (v1.6-23-gf0210b3). DNA variants were annotated by eVai v2.5 (EnGenome). Variants mapping in genes associated to the following Human Phenotype Ontology (HPO) phenotypes were prioritized and then filtered by MAF < 0.01 (GnomAD v2.1): HP0001249, 0001256, 0002187, 0002342, 0006887, 0006889, 0010864, 0012759. Filtered variants were classified according to ACMG-AMP criteria [8 (link)]. The most phenotype-fitting variant was confirmed by Sanger sequencing. Genomic DNA from both parents was analyzed for segregation analysis of the variant.
9
FFPE RNA Extraction and Fusion Gene Detection
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Total RNA were extracted from tumor containing spindle and epithelioid components in 5-μm thick formalin-fixed, paraffin-embedded (FFPE) unstained slides using the QIAamp DNA FFPE Kit (Qiagen, Hilden, Germany). The NGS platform used for the detection of fusion genes consists of Archer ® FusionPlex ® Pan Solid Tumor Panel (Diagnostica Longwood, Zaragoza, Spain), and NextSeq 550Dx sequencer (Illumina, San Diego, CA, USA). Sequencing data were analyzed with Archer Analysis 6.0 software (ArcherDX, Boulder, CO, USA).
10
Cell-free DNA Library Preparation for mNGS
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The size distribution of cfDNA was more narrow than that of cellular DNA. cfDNA is approximately 150–200 bp [20] and does not need to be sheared during library preparation. Cellular DNA is mainly genomic DNA, which must be sheared during library preparation. Therefore, different library preparation kits were selected for the two mNGS assays. NGS libraries of cfDNA were prepared using the KAPA DNA HyperPrep Kit (KK8504; Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s protocol. Cellular DNA libraries were constructed with the Illumina® DNA Prep (M) Tagmentation kit (20018705; Illumina, San Diego, USA). Library concentration was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the libraries were mixed in equimolar amounts. A 75-bp single-end sequencing was performed with a NextSeq™ 550Dx sequencer (Illumina, San Diego, USA), with at least 20 million sequencing reads obtained for each sample.
The amount of input DNA in mNGS test samples varied over six logs, from approximately 100 pg/mL in cell-poor body fluids such as cerebrospinal fluid to 100 μg/mL in cell-rich body fluids such as purulent discharge. The median read depth was 20 million (range: 3–22 million; interquartile range: 15–22 million). The turnaround time for the mNGS was approximately 24 h.
The amount of input DNA in mNGS test samples varied over six logs, from approximately 100 pg/mL in cell-poor body fluids such as cerebrospinal fluid to 100 μg/mL in cell-rich body fluids such as purulent discharge. The median read depth was 20 million (range: 3–22 million; interquartile range: 15–22 million). The turnaround time for the mNGS was approximately 24 h.
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