All samples were sorted using the Illumina HiSeq 4000 platform (Illumina, Inc., United states). A paired‐end library was constructed using 500 bp as the insert of each sample. The quality of the libraries of each sample was evaluated using DNA LabChip 1000 Kit and Agilent Bioanalyzer (Agilent Technologies, United Kingdom). The Illumina raw reads were screened as follows: 1) removal of reads containing three ambiguous N bases; 2) trim the reads containing low‐quality (Q < 20) bases; and 3) delete the reads containing< 60% high‐quality bases (Phred score ≥20). Then, a SOAPaligner (version 2.21) was used for the alignment of the clean reads to the National Center for Biotechnology Information GenBank bacterial genomes.
Dna labchip 1000 kit
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
The DNA LabChip 1000 kit is a microfluidic-based electrophoresis system designed for the analysis of DNA samples. The kit provides a standardized and automated method for sizing and quantifying DNA fragments ranging from 100 to 5,000 base pairs. The system uses a series of microchannels and detection electrodes to separate and analyze DNA samples.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (14 mentions)
Truseq dna sample prep v2 guide, by Illumina (7 mentions)
Agilent bioanalyzer, by Agilent Technologies (7 mentions)
Hiseq 2500, by Illumina (4 mentions)
Bioanalyzer, by Agilent Technologies (4 mentions)
Hiseq 4000 platform, by Illumina (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Qiaamp dna stool mini kit, by Qiagen (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Agilent 2100 expert software, by Agilent Technologies (2 mentions)
Multiplex pcr approach, by Toyobo (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Bioanalyser, by Agilent Technologies (1 mentions)
Dna stool kit, by Qiagen (1 mentions)
Nebnext ultra dna library prep kit, by Illumina (1 mentions)
Truseq sbs kit v3 hs, by Illumina (1 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Easypure stool genomic dna kit, by Transgene (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
30 protocols using dna labchip 1000 kit
1
Illumina Sequencing and Genome Alignment
2
Metagenomic DNA Sequencing from Fecal Samples
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DNA was extracted from faecal samples using the QIAampDNA Stool MiniKit as previously described10 (link). The metagenomic DNA libraries were constructed with 2 μg genome DNA according to the Illumina TruSeq DNA Sample Prep v2 Guide, with an average of 500 bp insert size. The quality of all libraries was evaluated using an Agilent bioanalyser with a DNA LabChip 1000 kit. Sequencing was performed by Illumina Hiseq2500.
3
Metagenomic DNA Library Construction Protocol
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The metagenomic DNA libraries were constructed with 2 μg genome DNA according to the IlluminaTruSeq DNA Sample Prep v2 Guide, with an average of 350 bp insert size (Qin et al., 2014 (link)). The quality of DNA libraries was evaluated via Agilent bioanalyzer with a DNA LabChip 1000 kit. Sequencing was carried out in Illumina Hiseq2500. Three replicates were performed.
4
Metagenomic DNA Extraction and Sequencing from Meconium
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In the laboratory, the meconium was divided into 5 aliquots (each 200-mg) and immediately stored at −80 °C. A frozen aliquot (200 mg) of each fecal sample was processed using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) for DNA extraction as previously described43 (link). The DNA concentration was measured with a NanoDrop (Thermo Scientific), and the molecular size was estimated by agarose gel electrophoresis.
Metagenomic DNA libraries were constructed with 0.2 μg of genomic DNA according to the Illumina TruSeq DNA Sample Prep v2 Guide, with an average insert size of 500 bp. The quality of all libraries was evaluated using an Agilent Bioanalyzer with a DNA LabChip 1000 kit. Negative controls (sterile water) were included for all the experimental process and showed no amplification in the final DNA library. Sequencing was performed using an Illumina Hiseq2500.
Metagenomic DNA libraries were constructed with 0.2 μg of genomic DNA according to the Illumina TruSeq DNA Sample Prep v2 Guide, with an average insert size of 500 bp. The quality of all libraries was evaluated using an Agilent Bioanalyzer with a DNA LabChip 1000 kit. Negative controls (sterile water) were included for all the experimental process and showed no amplification in the final DNA library. Sequencing was performed using an Illumina Hiseq2500.
5
Illumina TruSeq Metagenomic DNA Libraries
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The metagenomic DNA libraries were constructed with 2 μg genome DNA according to the Illumina TruSeq DNA Sample Prep v2 Guide, with an average of 350 bp insert size. The quality of all libraries was evaluated using an Agilent bioanalyser with a DNA LabChip 1000 kit. Sequencing was performed by Illumina Hiseq2500 at WuXi AppTec of China.
6
Illumina TruSeq DNA Library Prep for Gut Microbiome Analysis
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Following the Illumina TruSeq DNA Sample Prep v2 Guide (Illumina, Inc., San Diego, CA, USA), we constructed the DNA paired-end libraries with an insert size of 500 bp for the 76 fecal samples (24 from untreated active BD patients and 52 from normal controls). The quality of all libraries was evaluated using an Agilent bioanalyzer (Agilent Technologies, Wokingham, UK) and the DNA LabChip 1000 kit. All samples were subject to 150 bp paired-end sequencing on an Illumina HiSeq 4000 platform (Illumina, Inc., San Diego, CA, USA).
Raw reads were filtered to trim nucleotides from the 3′ end using a quality threshold of 30 and remove adaptor contamination and low-quality reads (e.g., reads containing more than 50% nucleotides below Q30, reads short than 70 bp, and reads mapped to the human genome based on alignment with SOAPaligner 2.21 [28 (link)]). As a consequence, an average of 95.82% high quality reads was obtained from all samples.
Raw reads were filtered to trim nucleotides from the 3′ end using a quality threshold of 30 and remove adaptor contamination and low-quality reads (e.g., reads containing more than 50% nucleotides below Q30, reads short than 70 bp, and reads mapped to the human genome based on alignment with SOAPaligner 2.21 [28 (link)]). As a consequence, an average of 95.82% high quality reads was obtained from all samples.
7
Metagenomic Shotgun Sequencing of Gut Microbiome
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Eight fecal samples were obtained from each of the eight mice in each group at the end of the 8-week intervention period. Bacterial chromatin was extracted using a DNA Stool kit (Qiagen Bioinformatics Co., Hilden, Germany) according to the manufacturer’s protocol. Subsequently, PCR was performed after ligating the adapters, size selection, and tailed random primers to obtain sufficient amplification products for library construction. The libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina, and their quality was validated with a DNA LabChip 1000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Wokingham, UK). Clusters were generated by bridge amplification within paired-end flow cells using the Illumina TruSeq PE Cluster Kit v3-cBot-HS, according to the manufacturer’s instructions. After cluster generation, paired-end sequencing was performed on an Illumina HiSeq 2000 platform using the TruSeq SBS Kit v3-HS. Unpaired reads were excluded from the clean reads. High-quality sequencing reads were de novo assembled into long contigs or scaffolds, which were used for gene prediction, taxonomic classification, and functional annotation. Detailed process for metagenomic shotgun sequencing was provided in online supplemental additional file 1 .
8
Fecal Metagenome DNA Extraction and Sequencing
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Genomic DNA from 69 fecal samples was extracted using the EasyPure® Stool Genomic DNA Kit (TransGen Biotech Co., Ltd, Beijing, China), and the metagenomic DNA library was constructed using the Nextera XT DNA Library Preparation Kit (Illumina, California, USA) following the manufacturer’s instructions. The quality of libraries was evaluated using an Agilent bioanalyser with a DNA LabChip 1000 Kit (Agilent, Palo Alto, USA). Samples with genomic DNA concentration less than 10 ng/μL, or abnormal quality (OD260/280>2.5, or OD260/280<1.5) were filtered out. Then, the cBOT was sequenced using the Illumina high-throughput sequencing platform NovaSeq6000 (Illumina, California, USA) according to the patent description provided by the Kangmeihuada Gene Technology Co., Ltd (Shenzhen, China) (20 ). After the low quality and ambiguous bases of the raw reads were filtered, the remaining reads were aligned to human genome reference (hg37) by KneadData to remove human host DNA contamination (21 (link), 22 (link)). The average rate of host contamination was (1.53 ± 2.18) %.
9
Metagenomic Sequencing Quality Evaluation
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The quality of all DNA libraries was evaluated using an Agilent bioanalyzer (Agilent Technologies, CA, USA) with the DNA LabChip 1000 kit. Whole-genome shotgun sequencing of PM samples collected was carried out on the Illumina Hiseq 4000 platform (Illumina, CA, USA) with 150-bp paired-end read length. In total, we obtained 946-Gb raw data (mean 8.8 Gb per sample, mean insert size 354 ± 83 bp). The raw reads of metagenomic sequencing were processed to remove low-quality reads and adaptor contaminations. Bases with a quality score < 30 were trimmed from 3′ end of reads, and reads < 70 bp were removed. Finally, we obtained 882-Gb clean data (mean 8.2 Gb per sample), and the proportion of high-quality reads was about 92.12% on average in all samples.
10
Metagenomic DNA Extraction and Sequencing from Brine Samples
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Total genomic DNA was extracted from brine samples using the E.Z.N.A.® Stool DNA Kit (D4015-02, Omega, Inc., United States) according to the manufacturer’s instructions. The metagenomic DNA libraries were generated with 2-μg genome DNA by applying the TruSeq™ DNA Sample Prep Kit (Illumina, San Diego, CA, United States). The average insert size is 350 bp. The quality of all libraries was assessed using an Agilent Bioanalyzer in combination with a DNA LabChip 1000 kit. Sequencing was conducted at LC-BIO TECHNOLOGIES CO., LTD. (Hangzhou, China) with an Illumina Genome Analyzer system pursuant to the manufacturer’s protocol. The sequencing data were submitted to NCBI Sequence Read Archive (SRA1 ) with accession number PRJNA780248 .
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