gDNA from three individuals (III‐1, III‐2, and IV‐1, Figure 1A ) from family GX‐052 was subjected to whole‐exome sequencing (WES) performed at Novogene Inc., Beijing, China. The Agilent SureSelect Human All Exon V6 kit and Illumina HiSeq PE150 (Illumina) were used to capture the targeted regions and sequence the DNA library at a mean coverage of 100X depth. The raw image files obtained from the HiSeq PE150 were processed with the Illumina pipeline for base calling, and then the clean reads were aligned to the human reference genome sequence (UCSC hg19 and NCBI database) by Burrows‐Wheeler Aligner (BWA) software, Samtools and GATK. The identified variants were analyzed according to the dominant inheritance model and filtered against public databases (the 1000 Genomes, Human Gene Mutation Database [HGMD], Exome Variant Server, and dbSNP databases). The Filter‐based annotation was performed according to a standard protocol.
Hiseq pe150
Manufactured by Illumina
Sourced in China, United States, Hong Kong, Singapore
The HiSeq PE150 is a high-throughput DNA sequencing system designed by Illumina. It performs paired-end sequencing with a read length of 150 base pairs. The system is capable of generating a large volume of sequencing data in a single run.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Nebnext ultra rna library prep kit for, by Illumina (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Plant genomic dna kit, by Tiangen Biotech (3 mentions)
Novaseq 6000 sequencing system, by Illumina (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
High sensitivity rna screentape, by Agilent Technologies (2 mentions)
Rna clean and concentrator with dnase 1 treatment, by Zymo Research (2 mentions)
Sureselect human all exon v6 kit, by Illumina (2 mentions)
Agilent 2100, by Agilent Technologies (2 mentions)
Plant dna extraction kit, by Tiangen Biotech (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Sureselect human all exon v6 kit, by Agilent Technologies (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
165 protocols using hiseq pe150
1
Whole-Exome Sequencing of Family GX-052
2
Mallard Genome Sequencing Workflow
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The blood samples were selected for DNA extraction using the phenol–chloroform protocol and the quality was detected by the Nanodrop ND-2000 spectrophotometer and observing DNA sample appearance. Two-end libraries of the evaluated samples were built according to the Illumina Hiseq PE150 platform. Small fragment libraries with a fragment length of 150 bp were constructed, and the successfully constructed libraries were sequenced via the Illumina Hiseq PE150 platform.
For quality assessment and detection of clean reads acquired from sequencing, low quality and adaptor sequences were filtered out by trinmomtic. The filtered high-qualified sequences were aligned to the mallard genome (https://asia.ensembl.org/Anas_platyrhynchos/Info/Index?db=core ) by BWA-mem, with the default alignment parameter for BWA-mem. The contrasted bam files were sorted using SAMtools and duplicate aligned sequences were removed using Picard. Sequences near indel were realigned using RealignerTargetCreator and IndelRealigner in GATK, while SNPs and indels within the genome were searched by Unified Genotyprer in GATK, and the SNPs and indels were filtered under: 1) QUAL > 30; 2) 18X > DP > 5X; 3) deletion rate < 0.1; 4) retained bialleles.
For quality assessment and detection of clean reads acquired from sequencing, low quality and adaptor sequences were filtered out by trinmomtic. The filtered high-qualified sequences were aligned to the mallard genome (
3
Whole Genome Sequencing of Plasmid-Containing E. coli
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Genomic DNA of E. 431, ECP.81 and ECP.82 were extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Plasmid DNA of ECP.81T and ECP.82T, the transconjugants obtained from ECP. 81 and ECP.82, were extracted by using Qiagen Plasmid Maxi Kit (Qiagen). The amount of all DNA samples was quantified according to Illumina sequencing sample requirements. The DNA samples were dissolved in 10mM Tris buffer to obtained at least 10 nM in 10 μl of minimum volume. The purity of DNA was determined at A260/280 and A260/230 using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Delaware, USA).
The quantified DNA were subjected to Whole Genome Sequencing (WGS) by using Illumina platform MiSeq or HiSeq PE150 (Illumina, San Diego, CA, US). The libraries were prepared by using Nextera XT sample preparation kit and sequenced with 2×250 or 350 paired-end reads protocol on an Illumina platform (MiSeq or HiSeq PE150) at Omics Sciences and Bioinformatics Center (OSBC), Faculty of Science, Chulalongkorn University and Singapore Joint Venture & Sequencing Center Novogen AIT.
The quantified DNA were subjected to Whole Genome Sequencing (WGS) by using Illumina platform MiSeq or HiSeq PE150 (Illumina, San Diego, CA, US). The libraries were prepared by using Nextera XT sample preparation kit and sequenced with 2×250 or 350 paired-end reads protocol on an Illumina platform (MiSeq or HiSeq PE150) at Omics Sciences and Bioinformatics Center (OSBC), Faculty of Science, Chulalongkorn University and Singapore Joint Venture & Sequencing Center Novogen AIT.
4
RNA-Seq Analysis of B-cells and Macrophages
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B-cells and iMO were sorted directly into Trizol. Total RNA was purified with the RNA Clean and Concentrator with DNase I treatment (Zymo Research), and RNA was eluted in 10mL. RNA purification was quantified by Qubit, and quality control was performed by RIN analysis (High Sensitivity RNA Screen Tape – Agilent) by the SKKC Metaomics Facility at Thomas Jefferson University. 1ng or 100pg of total RNA was used for library preparation with the mouse Ovation SoLo RNA-Seq Systems, including DNaseI and Insert-Dependent Adaptor Cleavage (InDA-C) treatments for DNA and rRNA depletion. Final libraries were run on Hiseq Illumina PE150 by Novogene. For B cell and iMO sorted from B6-Inf animals, three biological replicates of each population (Bys, Inf, and Naive) were sequenced, except for B cell Inf for which only two biological replicates were sequenced. For WT, Ifnar1−/− and Ifngr−/− iMO sorted from Inf BMCs, four biological replicates were sequenced for Bys and Inf WT iMO, and two biological replicates were sequenced for each Bys and Inf knockout iMO.
5
RNA-Seq Analysis of B-cells and Macrophages
6
Metagenomic Sequencing of Rumen Microbiome
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Total DNA was extracted from the frozen ruminal samples using a TIANamp Stool DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s protocols. The purity and the quality of the extracted DNA were assessed by electrophoresis and determined spectrophotometrically by measuring the A260/280 (Beckman DU/800; Beckman Coulter, Inc., Fullerton, CA, USA). Qualified DNA samples were randomly broken into approximately 350 bp fragments using Covaris M220 (Gene Company Limited, Woburn, MA, USA) for paired-end library construction and the whole library was prepared using a TruSeqTM DNA Sample Prep Kit (Illumina Inc., San Diego, CA, USA). Adapters were ligated to the blunt-end fragments, which contained the full complement of sequencing primer hybridization sites. Then, the resulting PCR products were purified and quantified on an Agilent Bioanalyzer 2100 system. Sample labeling was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS. Finally, paired-end sequencing was performed on an Illumina HiSeq PE150 (Illumina Inc., San Diego, CA, USA) platform according to the manufacturer’s instructions.
7
Transcriptome Sequencing of Brassica Cultivars
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Three experimental replicates from 9311 (ACK, AT1, AT1H1, AT3, and AT3H1) and DC907 (BCK, BT1, BT1H1, BT1H3, BT3, BT3H1, and BT3H3) cultivars were used for sequencing. Total RNA was extracted using an RNA Prep Pure Plant Kit (TIANGEN, Beijing, China) following the manufacturer’s protocol. RNA integrity was detected with an Agilent 2100 (Agilent Technologies, CA, USA). Oligo-dT primers and Array Script reverse transcriptase were used to construct the cDNA library. After library construction, initial quantification was performed using Qubit 2.0 to dilute the library to 1 ng/µL (Life Technologies, CA, USA), and then the insert size of the library was detected with Agilent 2100. After the insert size met expectations, the effective concentration of the library was quantified accurately by q-PCR (library effective concentration > 2 nM) to ensure library quality. All samples produced 150 paired terminal-sequence readings on the Illumina platform (Hiseq-PE150).
8
Transcriptome Profiling of Tumor and Spleen
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Corresponding tumor and spleen tissues were surgically removed from mice 5 days after the last treatment and preserved in 2 mL RNAlater (AM7021; Invitrogen) immediately. Total RNA was extracted with a TRIzol reagent (15596018; Ambion), then was used to prepare the eukaryotic strand-specific library and sequenced by an Illumina Hiseq-PE150.The data were normalized and analyzed with the “DESeq2” R package.
9
Lotus Genome Resequencing and Population Analysis
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A total of 240 lotus accessions were chosen for genome resequencing and population structure analyses. Sequencing libraries were generated using the TruSeq Nano DNA HT Sample Preparation Kit (Illumina), following the manufacturer’s instructions and were sequenced on the Illumina HiSeq PE150 platform. Low‐quality paired reads were filtered out of the data. The clean reads were mapped to the Asian lotus ‘China Antique’ reference genome using the burrows‐wheeler aligner (bwa ) and sorted bam files were obtained using samtools 1.9 (Li & Durbin, 2009 (link); Shi et al., 2020 (link)). sentieon (http://www.sentieon.com ) was used to identify the raw SNPs, and only high‐quality SNPs (coverage depth ≥ 4, RMS mapping quality ≥ 20, maf ≥ 0.05, miss ≤ 0.1) were retained for subsequent analysis. SNP annotation was performed using annovar (Wang et al., 2010 (link)). The neighbor‐joining (NJ) method based on the p‐distance using treebest 1.9.2 was used to construct an individual‐based phylogenetic tree that was visualized using mega 6 (Tamura et al., 2013 (link)). gcta was used to carry out principal component analysis (PCA) (Yang et al., 2011 (link)). admixture was used to perform an unsupervised cluster analysis, with the number of assumed genetic clusters K ranging from 2 to 5 (Alexander et al., 2009 (link)).
10
Mycobacterium Genome Sequencing and Assembly
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The 117 strains were cultured on Lowenstein Jensen media and genome DNA was extracted following the protocol of the cetyltrimethylammonium bromide (CTAB) method (22 (link)). Whole-genome sequencing was performed on the Illumina Hiseq PE150 platform by Annoroad (Beijing, China). The paired-end reads were examined using FastQC (v0.11.9) and trimmed using Trimmomatic (v0.39) (23 (link)). The genome sequences were assembled into a number of scaffolds by SPAdes (24 (link)). And the quality of the assemblies was evaluated using QUAST (v5.0.2).
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