For genomic sequencing and bioinformatic analyses, the bacterial DNA was extracted and purified using the Wizard® Genomic DNA Purification Kit (Promega). The quantity of the DNA was measured using a TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA). High quality DNA (OD260/280 = 1.8–2.0, > 1 μg) was used in sequencing library construction using the NEXTflex™ Rapid DNA-Seq Kit. The library was PCR amplified and used for paired-end Illumina sequencing (2 × 150 bp) on an Illumina HiSeq X Ten machine. Low quality data in the raw reads was removed to form clean data by a statistic of quality information for quality trimming, and the clean reads were assembled by SOAPdenovo v2.04. The inner gaps that emerged in the scaffold were filled using the Gap Closer version 1.12. The scaffolds were then uploaded to the CGView Server to plot the graphical circular genome map [75 (link)]. The coding sequences (CDS) were predicted using Glimmer version 3.02 and annotated from the Clusters of Orthologus Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases [76 (link)] using sequence alignment tools such as BLAST, Diamond and HMMER. tRNA and rRNA sequences were predicted by tRNA-scan-SE (v1.2.1) [77 (link)] and RNAmmer (v1.2) [78 (link)], respectively.
Hiseq x ten machine
Manufactured by Illumina
Sourced in United States
The HiSeq X Ten is a high-throughput DNA sequencing system developed by Illumina. It is designed to perform large-scale genome sequencing projects, with the ability to sequence up to 18,000 human genomes per year. The HiSeq X Ten utilizes Illumina's sequencing-by-synthesis technology to generate high-quality sequence data, making it a versatile tool for a variety of genomic applications.
Automatically generated - may contain errors
Lab products found in correlation
Wizard genomic dna purification kit, by Promega (3 mentions)
M220 focused acoustic shearer, by Covaris (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Qscript flex cdna synthesis kit, by Quanta Biosciences (2 mentions)
Direct zol rna kit, by Zymo Research (2 mentions)
Trizol reagent, by Merck Group (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Nextflextm rapid dna seq kit, by Illumina (2 mentions)
Nebnext ultra 2 rna library prep kit, by New England Biolabs (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Tbs 380 fluorometer, by Promega (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Micro kit v2, by Illumina (1 mentions)
Nextflextm rapid dna seq kit, by PSC Biotech (1 mentions)
Nextflex rapid dna seq kit, by PSC Biotech (1 mentions)
Pacbio sequel single molecule real time smrt, by Pacific Biosciences (1 mentions)
Purelink pro 96 genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
20 protocols using hiseq x ten machine
1
Bacterial Genome Sequencing and Annotation
2
Comprehensive TCRB Profiling in Murine Tumor Models
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Tumor, tumor-adjacent mammary mucosa, contralateral mammary mucosa, draining lymph nodes and spleen were isolated from 4T1 tumor bearing mice at 3-weeks post tumor injection and lysed in TRIzol Reagent (Sigma Aldrich). RNA was extracted using the Direct-zol RNA kit (Zymo Research) according to the manufacturer’s instructions. cDNA was synthesized using the qScript Flex cDNA synthesis kit (Quanta Biosciences) with a constant region specific primer (5′-ATCTCTGCTTCT- GATGGCTCA-3′). Multiplex PCR was performed to amplify the CDR3 region of rearranged TCRB loci and a set of primers, each specific to a specific TCR Vβ segments, and a reverse primer to the constant region of TCRB were used to generate a library of amplicons that cover the entire CDR3 region. PCR products were loaded on agarose gels and bands between 220–240 bp were extracted and purified using the QIAquick Gel Extraction kit (QIAGEN). These purified products were sequenced using the Illumina HiSeq X Ten machine.
3
Quantifying RNA Editing in Transcripts
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The cDNA was amplified with gene-specific primers flanking the target sequence (all primers and next-generation sequencing amplicons are listed in Supplementary Table S1 ). The A-to-I or C-to-U editing rates were evaluated from Sanger sequencing using the percentage of peak area at the target adenosine site according to the sequencing chromatograms. For deep sequencing of RNA amplicons, the cDNA was amplified with gene-specific primers with appropriate forward and reverse adaptor sequences. The PCR products were purified, and the amplicon libraries were constructed using the 300-cycle Mi-Seq Reagent Kit v2 or Micro Kit v2 according to the manufacturer's protocols for paired-end sequencing (2 × 150 bp) on the Illumina Hi-Seq X Ten machine by the Omics core facility of PICB. The RNA editing rates were evaluated by the percentage of the edited reads at the target sites (see below).
4
Comprehensive TCRB Profiling in Murine Tumor Models
5
Genomic DNA Extraction and Illumina Sequencing for Antimicrobial Resistance Analysis
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Strains exhibiting the same antimicrobial resistance patterns were excluded from sequencing analysis. After exclusion, 44 isolates were selected for sequencing. The genomic DNA of each isolate was extracted using a Wizard® Genomic DNA Purification Kit (Promega Corporation, Fitchburg, WI, United States) following the manufacturer’s protocol. DNA samples were sheared into 400- to 500-bp fragments using a Covaris M220 Focused-Ultrasonicator (ThermoFisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. Illumina sequencing libraries were then prepared from the sheared fragments using a NEXTflexTM Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, United States). Briefly, 5’ prime ends were first end repaired and phosphorylated. Next, 3’ ends were A-tailed and ligated to sequencing adapters. Adapter-ligated products were then enriched using PCR. Finally, the prepared libraries were used for paired-end Illumina sequencing (2 × 150 bp) on an Illumina HiSeq X Ten machine (Illumina, San Diego, CA, United States).
6
Hybrid genome sequencing protocol
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The genome was sequenced using a combination of PacBio Sequel Single Molecule Real Time (SMRT) (Pacific BioSciences, Menlo Park, MA, USA) and Illumina (Illumina, San Diego, CA, USA) sequencing platforms (Illumina, San Diego, CA, USA). For Illumina sequencing, at least 5 μg genomic DNA was utilized for sequencing library construction for each strain. The DNA samples were fragmented into 400–500 bp fragments using a Covaris M220 Focused Acoustic Shearer (Covaris, Woburn, MA, USA). The sheared fragments were subsequently utilized to generate Illumina sequencing libraries using the NEXTflex™ Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA). The 5′ ends of the fragments were first end-repaired and phosphorylated, while the 3′ ends were A-tailed and ligated to sequencing adapters. The adapter-ligated products were subsequently enriched through PCR. Subsequently, the prepared libraries underwent paired-end Illumina sequencing (2 × 150 bp) using the Illumina HiSeq X Ten machine (Illumina, San Diego, CA, USA).
The aliquot of 8 μg DNA was spun at 6000 rpm/min for 60 s. The DNA fragments were purified and end-repaired. The resulting sequencing library was purified three times using 0.45 times the volume of Agencourt AMPure XP beads (Beckman Coulter Genomics, Brea, MA, USA). Finally, an ~10 kb insert library was prepared and sequenced.
The aliquot of 8 μg DNA was spun at 6000 rpm/min for 60 s. The DNA fragments were purified and end-repaired. The resulting sequencing library was purified three times using 0.45 times the volume of Agencourt AMPure XP beads (Beckman Coulter Genomics, Brea, MA, USA). Finally, an ~10 kb insert library was prepared and sequenced.
7
Multiplex PCR and Sequencing of Genomic DNA
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Whole blood samples (10 mL) were collected from the patients and the peripheral blood mononuclear cells (PBMCs) were immediately isolated with Ficoll-Paque (GE Healthcare, Boston, USA) according to the manufacturer’s instructions. Genomic DNA was extracted from the PBMCs with a PureLink Pro 96 Genomic DNA Purification Kit (Invitrogen, California, USA) and used as a template for multiplex PCR with a Multiplex PCR Kit (Qiagen, Dusseldorf, Germany). The details of the PCR primers are shown in Table 1 . The Multiplex PCR protocol was as follows: pre-denaturation at 95°C for 15 min; 30 cycles at 94°C for 30 s, 60°C for 90 s, and 72°C for 30 s; final extension at 72°C for 5 min. Multiplex PCR amplification was done to construct the sequence library and the library was test to qualified for machine sequencing. High-throughput sequencing of each captured library ensured that the sequencing data volumes met the requirements. The amplicons were gel-extracted and purified prior to library preparation and the samples were sequenced as 150-bp paired-end runs on a HiSeq™ Xten machine (Illumina, California, USA).
8
Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). The genome was sequenced using a combination of PacBio RS II Single MoleculeReal Time (SMRT, Singapore, Singapore) and Illumina sequencing platforms. Illumina sequencing libraries were prepared from the sheared fragments using the NEXTflex™ Rapid DNA-Seq Kit. The prepared libraries then were used for paired-end Illumina sequencing (2 × 150 bp) on an Illumina HiSeq X Ten machine.
9
3' mRNA Sequencing with Lexogen
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QuantSeq 3′ mRNA‐Seq Library Prep Kit REV for Illumina (Lexogen) was used to sequence the 3′ end of RNA with polyA tail. In brief, 500 ng total RNA was taken as input. RNA 3′ end regions were reverse‐transcribed using an anchored oligo‐dT primer, and second strand synthesis was initiated by random priming. PCR amplification was then performed to obtain an Illumina compatible sequencing library. All libraries were sequenced in paired‐end 2 × 151 nt format on an Illumina HiSeq X Ten machine.
10
Targeted Sequencing of Eye Disease Genes
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For sequencing, 1 μg of the DNA sample from the proband of each family was sheared into fragments that were 200–500 bp long. The sheared fragments received blunt-end repair, and Klenow exonuclease was used to add a single-adenine base to the 3′ ends. Then, adapters (Illumina, San Diego, CA) were ligated to the repaired ends, and the DNA fragments were amplified in a PCR after ligation. PCR conditions were: denaturing at 98 °C for 2 min followed by 8 cycles of 98 °C for 30 s, 65 ℃ for 30 s and 72 °C for 1 min, then a final extension step at 72 °C for 10 min. The targeted DNA was captured using a customized panel of 762 genes, which included all the known genes related to eye diseases and sequenced by the Illumina HiSeq X Ten machine [9 (link)]. All the genes we detected are listed in Appendix 1.
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