Hiseq pe250 sequencing platform

Manufactured by Illumina

Sourced in China, United States

The HiSeq PE250 is a high-throughput sequencing platform designed for large-scale genomic studies. It uses paired-end sequencing technology to generate high-quality DNA sequence data with read lengths of up to 250 base pairs. The platform is capable of processing multiple samples simultaneously, making it suitable for a wide range of applications, including whole-genome sequencing, transcriptome analysis, and targeted sequencing.

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Lab products found in correlation

Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions) Axyprep dna gel extraction kit, by Corning (1 mentions) Primescript 2 1st strand cdna synthesis kit, by Takara Bio (1 mentions) Rnase free dnase 1 set, by Omega Bio-Tek (1 mentions) Qiaamp dna stool mini kit, by Qiagen (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)

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HiSeq platform (3071 citations) Illumina sequencing platform (388 citations) HiSeq sequencing platform (231 citations) HiSeq sequencing (173 citations) HiSeq PE250 (34 citations) PE250 (26 citations) HiSeq PE250 platform (20 citations) PE250 platform (19 citations)

6 protocols using hiseq pe250 sequencing platform

Fecal DNA Extraction and Sequencing Protocol

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Patients were asked to empty their bladder, then deliver stool into a sterile container. Approximately 2 g of feces were taken from the central part of the stool that had no contact with the air and placed in sterile cryotubes for storage at −80°C.
The fecal DNA was extracted and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., MA). The V3-V4 hypervariable regions were amplified via polymer chain reaction (PCR) using the universal primers, forward (5′–3′): CCTACGGGRSGCAGCAG (341F) and reverse (5′–3′): GGACTACVVGGGTATCTAATC (806R). The amplicons were purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA) followed by library quantification using a Qubit^TM dsDNA BR Assay Kit (Thermo Fisher Scientific). Finally, the pooled amplicons were paired end sequenced (2 × 250 bp) on an Illumina HiSeq PE250 sequencing platform. The sequencing depth was 42,501 reads per sample.

Zhang X., Li N., Chen Q, & Qin H. (2021). Fecal Microbiota Transplantation Modulates the Gut Flora Favoring Patients With Functional Constipation. Frontiers in Microbiology, 12, 700718.

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Comprehensive Genomic Analysis of Cecal Bacteria

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The total genomic DNA of cecal bacteria was extracted by using a DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The integrity of DNA was assessed by agarose gel electrophoresis, and then the genomic DNA was used as a template for PCR amplification. The 16S RNA V3–V4 gene region was amplified by using the primers F341 and R806 (Sun et al., 2017 (link)). PCR amplification was carried out in a 25 μl reaction system and the condition of PCR amplification was: initial pre-denaturation at 94°C for 4 min, denaturation at 98°C for 10 s, renaturation at 58°C for 30 s, elongation at 72°C for 2 min, 30 cycles, and then the last elongation step at 72°C for 10 min. The 16S rRNA gene was sequenced on the Illumina HiSeq PE250 sequencing platform at the Realbio Genomics Institute (Shanghai, China) according to the manufacturer’s instructions.

Liang H., Dai Z., Liu N., Ji Y., Chen J., Zhang Y., Yang Y., Li J., Wu Z, & Wu G. (2018). Dietary L-Tryptophan Modulates the Structural and Functional Composition of the Intestinal Microbiome in Weaned Piglets. Frontiers in Microbiology, 9, 1736.

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Soil Microbiome Profiling by 16S rRNA Sequencing

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Soil total RNA was extracted with E.Z.N.A. Soil RNA Mini Kits and purified with E.Z.N.A. RNase-Free DNase I Set according to the manufacturer’s protocols (Omega Bio-tek, Norcross, GA, USA). After extraction, RNA was used in reverse transcription to generate complimentary DNA (cDNA) by PrimeScript II 1st strand cDNA Synthesis Kits (Takara, Dalian, China). 16S cDNA was amplified by primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907R (5′-CCGTCAATTCCTTTGAGTTT-3′) [34 (link)]. PCR conditions were as follows: 98 °C for 1 min; 30 cycles at 98 °C (10 s), 50 °C (30 s) and 72 °C (30 s); and 72 °C for 5 min. Amplified PCR products were sequenced on an Illumina HiSeq PE250 sequencing platform (Illumina, San Diego, CA, USA). Sequences were clustered into operational taxonomic units (OTUs) with VSEARCH-2.11.1 [35 (link)] with a sequence similarity threshold of 0.97. Ribosomal database project training set v16 [36 (link)] was used for taxonomy annotations at a threshold of 0.8 using the SINTAX algorithm [37 ]. All sequence data were deposited into Genome Sequence Archive under PRJCA003001 and PRJCA003009.

Zhao K., Ma B., Xu Y., Stirling E, & Xu J. (2021). Light exposure mediates circadian rhythms of rhizosphere microbial communities. The ISME Journal, 15(9), 2655-2664.

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Rumen Microbiome DNA Extraction and Sequencing

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After thawing ruminal fluid, total DNA was extracted using the QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. The NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA) was used to measure quality and quantity of DNA samples. The V3–V4 regions of 16S rRNA gene were amplified using the 341F/806R primer set (5′-CCTAYGGGRBGCASCAG-3′/5′-GGACTACNNGGGTATCTAAT-3′) according to Xue et al. (72 (link)). Subsequently, amplicons were purified by QIAquick gel extraction kit (Qiagen, Hilden, Germany). The Illumina Hiseq-PE 250 sequencing platform (SanDiego, USA) was used to conduct amplicon sequencing by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The raw in vivo and in vitro amplicon sequence data generated in the study are available at the NCBI sequence read archive (SRA) under the accession number PRJNA666225 and PRJNA666235, respectively.

Ouyang J., Wang M., Bu D., Ma L., Liu F., Xue C., Du C., Aboragah A, & Loor J.J. (2021). Ruminal Microbes Exhibit a Robust Circadian Rhythm and Are Sensitive to Melatonin. Frontiers in Nutrition, 8, 760578.

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Bacterial DNA Extraction and 16S Sequencing

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Bacterial genomic DNA was extracted from nasal swab samples using a DNA Extraction Magnetic Bead Kit (Shenzhen BioEasy Biotechnologies Co., Ltd, China) according to the manufacturer's instructions and as previously described [21 (link)]. For 16S amplicon sequencing, PCR amplification spanning the bacterial V4-16S rRNA was performed [22 (link)], and the PCR products were mixed at a certain ratio by a Qubit fluorometer (Invitrogen™). The Illumina HiSeq PE250 sequencing platform was used for further sequencing. The raw 16S rRNA gene sequencing datasets are available in the European Bioinformatics Institute database (http://www.ebi.ac.uk/) under accession number PRJEB27877.

Tan X.L., Liu H.Y., Long J., Jiang Z., Luo Y., Zhao X., Cai S., Zhong X., Cen Z., Su J, & Zhou H. (2019). Septic patients in the intensive care unit present different nasal microbiotas. Future Microbiology, 14(5), 383-395.

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Gut Microbiome Analysis in Laying Hens

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Bacterial genomic DNA (gDNA) was extracted from the cecal digesta of laying hens using the PSP Spin Stool DNA Plus Kit (Invitek GmbH, Berlin, Germany) following the manufacturer's instructions. The quality of the extracted gDNA was assessed by gel electrophoresis. The V3–V4 region of the 16S rRNA gene was PCR-amplified using the 341F (5′-ACTCCTACGGGRSGCAGCAG-3′) and 806R (5′-GGACTACVVGGGTATCTAATC-3′) primer pair. Paired-end sequencing was performed on an Illumina Hiseq-PE250 sequencing platform at the Institute of Microbiology, Chinese Academy of Sciences (Beijing, China). The generated raw data were denoised, merged, and clustered using DADA2. Amplicon Sequence Variants (ASVs) were clustered with 100% similarity. Species annotation was performed for each ASV using the Naive Bayes classifier method based on the SILVA database. The phylum and genus composition of the microorganisms was visualized using the ggplot package in R. The alpha-diversity of the gut microbiota was calculated using the vegan package for R. Principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) were used to explore differences in community structure. Permutational multivariate analysis of variance (PERMANOVA) was used to test the statistical significance of the two principal components obtained from the PCoA and NMDS models.

Wei F., Yang X., Zhang M., Xu C., Hu Y, & Liu D. (2022). Akkermansia muciniphila Enhances Egg Quality and the Lipid Profile of Egg Yolk by Improving Lipid Metabolism. Frontiers in Microbiology, 13, 927245.

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