Hiseq4000

Manufactured by Pacific Biosciences

Sourced in United States

The HiSeq4000 is a high-throughput sequencing system designed for researchers. It is capable of generating large amounts of sequencing data for a wide range of applications.

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Lab products found in correlation

Rs 2 sequencing platform, by Pacific Biosciences (1 mentions) Qiaseq fx dna library kit, by Qiagen (1 mentions) Nanodrop 2000, by Thermo Fisher Scientific (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Pacbio rs 2, by Pacific Biosciences (1 mentions) Dna sample prep kit, by Illumina (1 mentions) Gentra puregene blood kit, by Qiagen (1 mentions) Biodrop mlite, by Harvard Bioscience (1 mentions) Rs 2, by Pacific Biosciences (1 mentions)

8 protocols using hiseq4000

Genome sequencing of strain WZN-1

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The genomic DNA of strain WZN-1 was extracted as described above in “Bacterial identification” section. The complete genome sequencing of WZN-1 was finished using Illumina Hiseq 4000 and PacBio RSII sequencing platform. The detailed sequencing information were described in a previous study (Wu et al. 2017 (link)). The specific degradation genes were identified and searched from the annotation of all the genes in WZN-1 use key words (such as dehalo, ring-opening) for further real-time qPCR analysis.

Wu Z., Xie M., Li Y., Gao G., Bartlam M, & Wang Y. (2018). Biodegradation of decabromodiphenyl ether (BDE 209) by a newly isolated bacterium from an e-waste recycling area. AMB Express, 8, 27.

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Fungal Genomic Library Construction and Sequencing

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Fungal strains were grown in potato dextrose broth for 1 wk and conidia were harvested for DNA isolation according to Epstein et al (25 (link)). Genomic libraries were constructed using the QIA Seq FX DNA library kit according to instructions from Qiagen Inc. Sequencing was performed on either the Illumina HiSeq 4000 or the PacBio Sequel II HiFi platform at the University of California Davis, Genome Center. Illumina sequence data were assessed using FastQC for per base and sequence quality score, GC content, and sequence length distribution. Low-quality reads were removed and adapters were trimmed using Trimmomatic v36 (65 (link)), and error correction was performed using ALLPATHS-LG (66 (link)). Draft genome assemblies were obtained using A5 (67 (link)). Genome completeness was assessed using the Sordariomyceta odb9 set of BUSCO (v3) (38 ). Contigs representing potential contaminant data were identified using FCS-GX, developed by the National Center for Biotechnology Information and available with documentation at https://github.com/ncbi/fcs/wiki/FCS-GX. Putative contaminant contigs were removed, except in cases where pangenome orthology searches identified bonafide (>95% identity) F. oxysporum genes. PacBio data were assembled using Hifiasm v0.16.0 using the program’s default parameters (68 (link)).

Fayyaz A., Robinson G., Chang P.L., Bekele D., Yimer S., Carrasquilla-Garcia N., Negash K., Surendrarao A., von Wettberg E.J., Kemal S.A., Tesfaye K., Fikre A., Farmer A.D, & Cook D.R. (2023). Hiding in plain sight: Genome-wide recombination and a dynamic accessory genome drive diversity in Fusarium oxysporum f.sp. ciceris. Proceedings of the National Academy of Sciences of the United States of America, 120(27), e2220570120.

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Olympia Oyster Genome Assembly

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To facilitate the analysis of genetic and epigenetic data, a draft genome for the Olympia oyster was developed using a combination of short-read sequence data (Illumina HiSeq4000) combined with long-read sequence data (PacBio RSII) using PBJelly (PBSuite_15.8.24; English et al, 2012). Short reads (NCBI SRA: SRP072461) were assembled using SOAPdenovo (Li et al, 2008) . The scaffolds (n=765,755) from this assembly were combined with the PacBio longread data (NCBI SRA: SRR5809355) using PBJelly (PBSuite_15.8.24; English et al, 2012).
Assembly with PBJelly was performed using the default settings. Only contigs longer than 1000 bp were used for further analysis. Genome assembly parameters were compiled using QUAST (v4.5; Gurevich et al, 2013).
Genome annotation was performed using MAKER (v.

Silliman K., Spencer L.H., White S.J., & Roberts S. (2022). Epigenetic and genetic population structure is coupled in a marine invertebrate.

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Comprehensive RNA Extraction and Sequencing

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The M and N portions of the leaves were ground into powder in liquid nitrogen, respectively. Total RNA extractions were done using the RNAprep Pure Plant Kit (DP441, Tiangen, China) following the manufacturer’s instructions. RNA integrity and quantity were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA), making sure all the RNA integrity numbers (RIN) were at least 8.0. RNA concentration was measured using Nanodrop 2000 (Thermo Fisher Scientific, USA). To ensure obtaining enough RNA for sequencing, each RNA sample was mixed from five individual plant leaves. RNA extraction, DNA library construction, and transcriptome sequencing were completed by Biomarker Technologies Corporation (Beijing, China) using Illumina HiSeq4000 (next-generation sequencing, NGS) and Pacific Biosciences (PacBio) Sequel II (third-generation sequencing, TGS) platform. Quality checks of the cDNA libraries were done by Qubit (> 20 ng/μL), and Agilent 2100 (fragment range 250–390 bp, without impurity peaks).

Huang H., Zou H., Lin H., Dai Y, & Lin J. (2023). Molecular insights into the mechanisms of a leaf color mutant in Anoectochilus roxburghii by gene mapping and transcriptome profiling based on PacBio Sequel II. Scientific Reports, 13, 22751.

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Genome Sequencing and Assembly Protocol

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High-purity genomic DNA was extracted by successive phenol/chloroform/isoamyl alcohol purification steps followed by precipitation with 2-propanol, treatment with RNase, and a final purification and precipitation step.¹⁷ Quantification was done in 1% agarose gel electrophoresis with a NanoDrop spectrometer (Saveen Werner, Sweden) and Qubit 2.0 analyzer (Invitrogen, UK). Genome sequencing was performed using Illumina HiSeq4000 and Pacific Biosciences RS single-molecule real-time (SMRT) sequencing platforms at the IGM Genomics Center, University of California, San Diego, La Jolla, CA, USA. Genome assembly was done with the hybridSPAdes algorithm.^{18 (link)} Final polishing steps (correction of ambiguous bases and circularization) were performed using PCR and Sanger sequencing. The genome was submitted to the National Center for Biotechnology Information (NCBI) database under the accession number CP024087. Biosynthetic gene clusters were identified in the assembled genome using AntiSMASH 4,¹⁹ and domains identified in the neaumycin gene cluster aligned using CLC Main Workbench version 7.8.1 (gap open cost: 10, gap extension cost: 1, end gap cost: as any other).

Kim M.C., Machado H., Jang K.H., Trzoss L., Jensen P.R, & Fenical W. (2018). Integration of Genomic Data with NMR Analysis Enables Assignment of the Full Stereostructure of Neaumycin B, a Potent Inhibitor of Glioblastoma from a Marine-Derived Micromonospora. Journal of the American Chemical Society, 140(34), 10775-10784.

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Whole Blood Genomic DNA Extraction and Sequencing

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We used a Gentra Puregene Blood Kit (Qiagen) to extract the genomic DNA from a whole blood sample according to the manufacturer’s instructions. Then we assessed the quality of DNA by electrophoresis on 1% agarose gel and the quantity of DNA by a BioDrop mLITE pectrophotometer (a total of 15 mg of DNA was quantified using the spectrophotometer). The sequencing included two platforms: Illumina (San Diego, CA) Hiseq4000 and Pacific Biosciences (Menlo Park, CA) PacBio RSII. We used Illumina DNA Sample Prep Kit to construct three paired-end libraries with insert size of 350 bp and SMRT bell library preparation protocol (10 SMRT cells were sequenced) to generated by Pacific Biosciences long reads (> 10 kb).

Song K., Gao B., Halvarsson P., Fang Y., Jiang Y.X., Sun Y.H, & Höglund J. (2020). Genomic analysis of demographic history and ecological niche modeling in the endangered Chinese Grouse Tetrastes sewerzowi. BMC Genomics, 21, 581.

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Isolation and Characterization of C. lupini

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The RB221 strain of C. lupini (IMI 504893; UBOCC-A-117274) was isolated from a lupin crop in Brittany in 2014. The genome sequence of this strain was obtained using Illumina GAII and HiSeq 4000 instruments, and also using Pacific Biosciences technology (PacBio, Menlo Park, CA, USA). The inoculum of the strain RB221 was prepared from a two week-old PDA culture at 25 °C, by adding 1.5 mL of distilled sterile water to the culture. Spore concentration was adjusted to 1 × 10⁴ spores·mL⁻¹. Five microliters of the spore suspension was used to inoculate sterilized lupin boiled seeds (adapted from Saubeau et al. (2014) [35 (link)]) placed on 1.3% water-agar Petri plates. After one-week of incubation at 25 °C, spores were harvested by shaking the infected lupin seeds into 3–4 mL of distilled water, and the spore inoculum was adjusted to 1 × 10⁷ spores·mL⁻¹. For each biological repetition, a new fungal culture and inoculum was prepared.

Dubrulle G., Picot A., Madec S., Corre E., Pawtowski A., Baroncelli R., Zivy M., Balliau T., Le Floch G, & Pensec F. (2020). Deciphering the Infectious Process of Colletotrichum lupini in Lupin through Transcriptomic and Proteomic Analysis. Microorganisms, 8(10), 1621.

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Genomic DNA Isolation of S. flexneri

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The representative S. flexneri strains were grown in lysogeny broth (LB) overnight, and their DNA was purified using a modified alkaline lysis and phenol-chloroform extraction method (82 (link)). The purified DNA was used to construct libraries and was sequenced on the Illumina HiSeq 4000 and the Pacific Biosciences RS II with P6C4 chemistry and were assembled as previously described (82 (link)).

Gabor C.E., Hazen T.H., Delaine-Elias B.C., Rasko D.A, & Barry E.M. (2023). Genomic, transcriptomic, and phenotypic differences among archetype Shigella flexneri strains of serotypes 2a, 3a, and 6. mSphere, 8(6), e00408-23.

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