The fecal samples were obtained from KM mice drinking 1% CS-A (w/v) for 3 months. The total genomic DNA was extracted, sonicated, and used to construct a Paired-end library with an average size of about 400 bp. The Illumina NovaSeq/Hiseq Xten (Illumina Inc) was used for sequencing. The quality of raw data was checked by fastp (https://github.com/OpenGene/fastp ) and then high-quality reads were acquired and assembled by Megahit (https://github.com/voutcn/megahit ) (45 (link)). The assembled contigs were predicted by MetaGene (http://metagene.cb.k.u-tokyo.ac.jp/ ) (46 (link)) and then the predicted proteins IM3796 and IM1634 were annotated by the Carbohydrate Active enZYme (CAZy) (http://www.cazy.org/ ) (38 (link), 47 (link)).
Novaseq hiseq xten
Manufactured by Illumina
Sourced in United States
The NovaSeq/HiSeq Xten is a next-generation sequencing system designed for high-throughput DNA sequencing. It is capable of generating large volumes of sequence data efficiently. The system utilizes advanced sequencing-by-synthesis technology to produce accurate and reliable DNA sequencing results.
Automatically generated - may contain errors
Lab products found in correlation
E z n a soil dna kit, by Omega Bio-Tek (7 mentions)
Hiseq x reagent kit, by Illumina (5 mentions)
Novaseq reagent kit, by Illumina (5 mentions)
Nextflextm rapid dna seq, by PSC Biotech (4 mentions)
Nextflex rapid dna seq, by PSC Biotech (3 mentions)
Novaseq reagent kits hiseq x reagent kits, by Illumina (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Matrix e beads, by MP Biomedicals (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Proteinase k, by Qiagen (1 mentions)
12 protocols using novaseq hiseq xten
1
Metagenomics Analysis of Gut Microbiome
2
Cecal Microbiota Profiling by Metagenomics
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Metagenome sequencing was used to investigate the cecal microbiota in greater detail, as previously described (41 (link)), with some modifications. To summarize, total genomic DNA was extracted from the cecal contents 26 of chickens SE-infected using the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, United States) according to the manufacturer’s protocol. TBS-380 and NanoDrop2000 were used to determine the concentration and purity of extracted DNA, respectively. On a 1% agarose gel, the DNA quality was determined. The extracted DNA was then fragmented to an average size of approximately 400 bp using the Covaris M220 (Gene Company Limited, China) to construct paired-end libraries. Using NEXTflexTM Rapid DNA-Seq, a paired-end library was built (Bioo Scientific, Austin, TX, USA). To the blunt end of the fragments, adapters containing the complete complement of sequencing primer hybridization sites were ligated. On the Illumina NovaSeq/Hiseq Xten, paired-end sequencing was performed (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (www.illumina.com ).
3
Gut Microbiome DNA Extraction and Sequencing
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Total genomic DNA was extracted from colon content samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. The concentration and purity of extracted DNA were determined with TBS-380 and NanoDrop2000, respectively. DNA extract quality was checked on 1% agarose gel.
DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, Hong Kong, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt end of fragments. Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (www.illumina.com , accessed on 10 December 2021). Sequence data associated with this project were deposited in the NCBI Short Read Archive database (Accession Number: PRJNA849732).
DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, Hong Kong, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt end of fragments. Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (
4
Microbial Genome Sequencing Protocol
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The DNA extract was fragmented to an average size of approximately 300 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end sequencing was performed on an Illumina NovaSeq/HiSeq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (www.illumina.com ). Reads were aligned to the mouse genome by BWA (http://bio-bwa.sourceforge.net ), and any hits associated with the reads and their mates were removed. All predicted genes with 95% sequence identity (90% coverage) were clustered using CD-HIT (http://www.bioinformatics.org/cd-hit/ ), and the longest sequences from each cluster were selected as representative sequences to construct a nonredundant gene catalog. After quality control, reads were mapped to the representative sequences with 95% identity using SOAPaligner (http://soap.genomics.org.cn/ ), and the gene abundance in each sample was evaluated. Read counts assigned to the fungal kingdom were then extracted for analysis.
5
Donkey Gut Microbiome DNA Extraction
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Total genomic DNA was extracted from 100 mg of frozen contents from the hindgut content (cecum, colon, ventral colon, and dorsal colon) of donkeys using an E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.) according to the manufacturer’s instructions. DNA yield and quality were determined with a NanoDrop2000 (Thermo Scientific, Wilmington, USA). DNA fragments with an average size of approximately 400 bp were sequenced on an Illumina NovaSeq/HiSeq XTen instrument (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (www.illumina.com ).
6
Shotgun Metagenomics of Dietary Fiber Modulation
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Digesta of the large intestine of both dietary fiber deprivation and xylan supplementation were subjected to shotgun metagenomics sequencing. Briefly, total genomic DNA was extracted using the E.Z.N.A Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.). Concentration and purity were determined with TBS-380 and NanoDrop2000, respectively. The quality of extracted DNA was checked on 1% agarose gel. Then, DNA was fragmented to approximately 400 bp using Covaris M220 (Gene Company Limited, China). Adapter ligation, cleanup, and enrichment were performed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, USA). Shotgun metagenomic sequencing was performed on Illumina NovaSeq/Hiseq Xten at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Quality control was performed using fastp (version 0.19.4) to trim adapters and filter low-quality reads with the parameter “--cut_by_quality3 -W 4 -M 20 -n 5 -c -l 50 -w 3” [27 (link)]. Bowtie2 (version 2.4.1) was used to remove reads aligned to the swine genome [28 (link)].
7
Metagenomic DNA Extraction and Sequencing
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Total genomic DNA was extracted from Colon content samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions. Concentration and purity of extracted DNA was determined with TBS-380 and NanoDrop2000, respectively. DNA extract quality was checked on 1% agarose gel.
DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt-end of fragments. Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions.1 Sequence data associated with this project have been deposited in the NCBI Short Read Archive database (Accession Number: PRJNA849732).
DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt-end of fragments. Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions.
8
Soil Microbial DNA Extraction and Sequencing
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The E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) was used to extract microbial DNA. Paired-end sequencing was performed using an Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) (Yang et al., 2021 ). The detailed protocol has been described previously (Liang et al., 2021 ).
9
Fecal DNA Isolation and Sequencing
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Total genomic DNA was extracted from human fecal samples as mentioned in 16S rRNA sequencing. DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end library was constructed using NEXTflexTM Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt-end of fragments. Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc, San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co, Ltd (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (https://www.illumina.com ).
10
Gut Microbiome Profiling Protocol
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Stomach microbiome samples (n = 10: 4 HPI and 6 uninfected) were bead-beaten with Matrix E beads (MP Biomedicals) for three 2-minute intervals and then incubated with lysozyme (100 mg/mL; Sigma-Aldrich) at 37°C for 60 minutes followed by digestion with 20 μL proteinase K (20 mg/mL; Qiagen) at 55°C and 250 rpm for 90 minutes. Data extraction steps were performed with DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s guidelines. Metagenomic sequencing and analysis were performed by Novogene using a standard protocol for low-input samples. Sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions.
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