Total RNA from tumor tissue was extracted using mirVana miRNA Isolation Kit (Thermo Fisher Scientific, MA, USA). Quantity and quality of extracted RNA were checked by Qubit® 2.0 Fluorometer system (Thermo Fisher Scientific, MA, USA) and NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, MA, USA). For sequencing libraries preparation, TruSight RNA Pan-Cancer Panel (Illumina, CA, USA), which targets fusions in 1385 genes, was used. Sequencing libraries were subsequently loaded on NextSeq 500/550 Mid Output Kit v2 (150 cycles) and NextSeq 500 sequencing device (both Illumina, CA, USA). All processes were performed according to the manufacturer's instructions. Quantity and quality of sequencing libraries were checked by Qubit® 2.0 Fluorometer system (Thermo Fisher Scientific, MA, USA) and TapeStation 2200 (Agilent Technologies, CA, USA). For data analysis, BreakingPoint tool was used.
Qubit 2.0 fluorometer system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit 2.0 fluorometer system is a compact and versatile instrument designed for quick and accurate quantification of DNA, RNA, and protein samples. The system uses fluorescence-based detection technology to provide precise measurements of sample concentrations. It is capable of analyzing a wide range of sample types and volumes, making it a useful tool for various applications in life science research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (6 mentions)
Nextflex 16s v1 v3 amplicon seq kit, by PerkinElmer (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Biospecnano spectrophotometer system, by Shimadzu (1 mentions)
Nextflex rapid dna seq kit, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Trusight one kit, by Illumina (1 mentions)
Variantstudio, by Illumina (1 mentions)
Nextera rapid capture panel, by Illumina (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Trusight rna pan cancer panel, by Illumina (1 mentions)
Nextseq 500 sequencing device, by Illumina (1 mentions)
Nextseq 500 550 mid output kit v2, by Illumina (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Truseq nano prep kit, by Illumina (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
11 protocols using qubit 2.0 fluorometer system
1
Tumor RNA Extraction and Sequencing
2
Evaluating Algorithm Performance on Contaminated Samples
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Algorithm performance was tested on samples contaminated artificially at different levels. To generate the contaminated samples, we used three Coriell samples, NA20828, NA20582 and NA20763. First, we measured the concentration of each sample using a BiospecNano Spectrophotometer system (Shimadzu Corporation, Japan) and we diluted them to a concentration of 20 ng/µl. Then, we measured again the concentration of each 20 ng/µl sample using a dsDNA BR Assay Kit on a Qubit 2.0 Fluorometer System (Invitrogen, Carlsbad, CA, USA) to have the most accurate value. At the end, we diluted the samples to the final desired concentration of 5 ng/µl in 10 µl final volume according to Illumina Nextera Rapid Capture Protocol. We prepared 5, 7 and 2 aliquots of NA20828, NA20582 and NA20763 at 5 ng/µl, respectively, to have enough material to combine for the contamination process. NA20828, NA20582 were used as principal samples and were contaminated at different levels with NA20582 and NA20763 respectively. A total of nine samples with known contaminations ranging from 2% to 20% were generated (Table 1 ). The algorithm was tested on 12 samples, the nine contaminated plus the three Coriell samples as controls. All the final samples were at 5 ng/µl in 10 µl volume.
3
NGS Library Preparation using NEXTflex
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The mixture of purified PCR products was generated for Next-generation sequencing (NGS) library using NEXTflex Rapid DNA-Seq kit for Illumina (BIOO SCIENTIFIC, USA) following manufacturer’s recommendations. The library quality was quantified by Qubit dsDNA HS Assay Kit with the Qubit 2.0 fluorometer system (Invitrogen, Life Technologies, Grand Island, NY, USA). The multiplexed amplicons were sequenced using the Illumina HiSeq. 2500 platform to generate 250 bp paired-end reads. The sequencing and analysis were performed at SinoGenoMax Co., Ltd, Beijing, China.
4
Metagenomic Sequencing Library Preparation
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The PCR products of 16S V4 (∼300 bp) and V6 (∼290 bp) region amplified from bacterial communities were purified by QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Purified PCR products were quantified by Qubit® dsDNA HS Assay Kit with the Qubit® 2.0 Fluorometer system (Invitrogen, Life Technologies, Grand Island, NY, USA). For each metagenomic sample, 16S V4 and V6 amplicons were directly used to construct paired-end (PE) libraries using NEBNext® Ultra™ DNA Library Prep Kit.
LD-IPCR products from circular AatII-digested genomic DNA and circular EcoRI-digested genomic DNA were sheared into fragments of ∼900 and ∼500 bp in size by sonication using Covaris s220 (Covaris, Woburn, MA, USA), respectively. PE libraries were constructed according to a standard protocol with NEBNext® Ultra™ DNA Library Prep Kit. Biotin-labeled LD-IPCR products were adsorbed onto magnetic streptavidin beads and then bound to the magnet for enrichment. Subsequently, NGS libraries of these biotinylated PCR fragment were performed the same as above.
Purified metagenomic DNA sequences were fragmented into ∼180 bp by sonication using Covaris s220 (Covaris). PE libraries were constructed according to a standard protocol provided by Illumina, Inc. (San Diego, CA, USA).
LD-IPCR products from circular AatII-digested genomic DNA and circular EcoRI-digested genomic DNA were sheared into fragments of ∼900 and ∼500 bp in size by sonication using Covaris s220 (Covaris, Woburn, MA, USA), respectively. PE libraries were constructed according to a standard protocol with NEBNext® Ultra™ DNA Library Prep Kit. Biotin-labeled LD-IPCR products were adsorbed onto magnetic streptavidin beads and then bound to the magnet for enrichment. Subsequently, NGS libraries of these biotinylated PCR fragment were performed the same as above.
Purified metagenomic DNA sequences were fragmented into ∼180 bp by sonication using Covaris s220 (Covaris). PE libraries were constructed according to a standard protocol provided by Illumina, Inc. (San Diego, CA, USA).
5
Next-Generation Sequencing of 16S V1-V3
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Library for next-generation sequencing (NGS) was generated with NEXTflex® 16S V1-V3 Amplicon-Seq Kit (PerkinElmer, catalog number NOVA-4202-04), according to the manufacturer's instructions. The library quality was quantified by Qubit dsDNA HS Assay Kit with the Qubit 2.0 fluorometer system (Invitrogen, Life Technologies, Grand Island, NY, USA). Amplicons were sequenced on the MiSeq instrument (Illumina).
6
Fecal DNA Extraction and Microbiome Analysis
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DNAs were isolated from fecal using the QIAamp DNA Mini Kit (Qiagen, 51306). The concentration of double-stranded DNA in isolated samples was determined using a Qubit 2.0 instrument and a Qubit dsDNA HS Assay kit (ThermoFisher, catalog number 32853).
Library for Next-generation sequencing (NGS) generated with NEXTflex® 16S V1-V3 Amplicon-Seq Kit (PerkinElmer, catalog number NOVA-4202-04), according to the manufacturer's instructions. The library quality was quantified by Qubit dsDNA HS Assay kit with the Qubit 2.0 fluorometer system (Invitrogen, Life Technologies, Grand Island, NY, USA). Amplicons were sequenced on the MiSeq instrument (Illumina Systems).
Demultiplexing, filtering, denoise, chimeric sequences, and determining OTU and taxonomic identification were performed using LotuS pipeline (Hildebrand et al. 2014) (link).
Analysis of alpha diversity to assess the abundance of the community, the calculation of alpha biodiversity (Shannon indices), beta biodiversity as well as the construction of taxonomic distribution at the phylum and genus level were performed using vegan (Oksanen et al. 2019 ) and phyloseq R packages (v.1.24.2) (McMurdie and Holmes 2013) and graphs were generated using web-based platform for comprehensive analysis -MicrobiomeAnalyst (Chong et al. 2020) (link).
Library for Next-generation sequencing (NGS) generated with NEXTflex® 16S V1-V3 Amplicon-Seq Kit (PerkinElmer, catalog number NOVA-4202-04), according to the manufacturer's instructions. The library quality was quantified by Qubit dsDNA HS Assay kit with the Qubit 2.0 fluorometer system (Invitrogen, Life Technologies, Grand Island, NY, USA). Amplicons were sequenced on the MiSeq instrument (Illumina Systems).
Demultiplexing, filtering, denoise, chimeric sequences, and determining OTU and taxonomic identification were performed using LotuS pipeline (Hildebrand et al. 2014) (link).
Analysis of alpha diversity to assess the abundance of the community, the calculation of alpha biodiversity (Shannon indices), beta biodiversity as well as the construction of taxonomic distribution at the phylum and genus level were performed using vegan (Oksanen et al. 2019 ) and phyloseq R packages (v.1.24.2) (McMurdie and Holmes 2013) and graphs were generated using web-based platform for comprehensive analysis -MicrobiomeAnalyst (Chong et al. 2020) (link).
7
Genetic Analysis of Channelopathy Genes
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All procedures were conducted as part of routine clinical care. The study was performed under the ethics guidelines issued by our institution, with written informed consent obtained from all participants for genetic studies.
Genetic analysis for PP genes SCN4A, CACNA1S, and KCNJ2 was performed at the Neurogenetics Unit, National Hospital for Neurology and Neurosurgery as provided by the Channelopathy Highly Specialized National Service for rare disease. Samples underwent next-generation sequencing on an Illumina HiSeq after enrichment with an Illumina custom Nextera Rapid Capture panel (Illumina, Inc, San Diego, CA). For case 2, the library preparation and enrichment were performed with TruSight One kit (Illumina) according to protocol instructions, allowing analysis of a panel of ≈5,000 genes (including PP genes and RYR1). The library was quantified with the Qubit 2.0 Fluorometer system (Thermo Fisher Scientific, Waltham, MA), and 2 × 250-bp paired-end sequencing was performed on the MiSeq sequencer (Illumina), as well as sequences alignment (Burrows-Wheeler aligner) and variant calling (Genome Analysis Toolkit variant caller). The variants were then analyzed with VariantStudio (Illumina).
Additional targeted RYR1 Sanger sequencing of all cases was performed at the Diagnostic DNA Laboratory at Guy's Hospital, London.
Genetic analysis for PP genes SCN4A, CACNA1S, and KCNJ2 was performed at the Neurogenetics Unit, National Hospital for Neurology and Neurosurgery as provided by the Channelopathy Highly Specialized National Service for rare disease. Samples underwent next-generation sequencing on an Illumina HiSeq after enrichment with an Illumina custom Nextera Rapid Capture panel (Illumina, Inc, San Diego, CA). For case 2, the library preparation and enrichment were performed with TruSight One kit (Illumina) according to protocol instructions, allowing analysis of a panel of ≈5,000 genes (including PP genes and RYR1). The library was quantified with the Qubit 2.0 Fluorometer system (Thermo Fisher Scientific, Waltham, MA), and 2 × 250-bp paired-end sequencing was performed on the MiSeq sequencer (Illumina), as well as sequences alignment (Burrows-Wheeler aligner) and variant calling (Genome Analysis Toolkit variant caller). The variants were then analyzed with VariantStudio (Illumina).
Additional targeted RYR1 Sanger sequencing of all cases was performed at the Diagnostic DNA Laboratory at Guy's Hospital, London.
8
Coyote DNA Extraction and Quantification
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We obtained 16 blood or tissue samples from coyote individuals in NYC and surrounding areas (Table S1 ), including a suspected pack near Elmjack Baseball Field in Queens (Figure 1 ). These samples were collected opportunistically from carcasses and animals trapped and euthanized/released by NY State and City officials. This work was conducted under the approved Princeton University IACUC protocol 1961A. We obtained high-molecular-weight genomic DNA using the Qiagen High-Molecular-Weight DNA Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol for enucleated whole blood and frozen tissue. We quantified DNA concentration using the Qubit 2.0 fluorometer system (Thermo Fisher Scientific, Carlsbad, CA, USA) and subsequently standardized each sample’s DNA concentration to 5 ng/µL.
9
Shotgun Sequencing of Middle Ear Effusion
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Quality control was performed on 49 DNA samples from middle ear effusion using the Qubit 2.0 fluorometer system (Life Technologies, Boston, MA) and the nanodrop. These samples were further processed for shotgun sequencing using a Truseq Nano prep kit (Illumina, San Diego, CA). Genomic libraries were validated (concentration and size) with the Qubit and 2100 Bioanalyzer. Forty-four samples passed the validation and were sequenced on the Nextseq 500 using the 2x151 basepair paired end protocol, after an initial loading titration trial sequencing run. Raw reads (FASTQ format) were preprocessed (QC) using PRINSEQ-lite 0.20.4 (trimming reads and bases < 25 PHRED, minimum length = 100, removing exact duplicates, reads with undetermined bases, and low complexity reads using Dust filter = 25)[30 (link)]. Filtered (paired and unpaired reads) were aligned to the human genome (hg19) using Bowtie2 (—very-sensitive)[31 (link)].
10
Evaluation of Amplicon Preparation Biases
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Two mock communities were used to evaluate amplicon preparation, data quality, and quantitative biases. Both communities consisted of the same five 16S rRNA gene clones (H42, AF234715; H29, AF234692; H28, AF234749; H13, AF234737; H44, AF234743) from an activated sludge study (Juretschko et al., 2002 (link)) that were combined to have even or uneven proportions. Purified plasmids were quantified using the Qubit® dsDNA BR Assay Kit (Life Technologies) and a Qubit® 2.0 Fluorometer system (Life Technologies). For the even mock community the plasmids were mixed in equimolar proportions, for the uneven mock community the clones were mixed in a more realistic fashion, resulting in relative abundances of the individual clones at 76, 18, 5, 0.7, and 0.09%, respectively. After construction, the even and uneven mock communities were diluted to a final concentration of 0.1 ng μL−1.
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