Whole genomic DNA from the LAB isolate was extracted using DNeasy Blood & Tissue Kits (Qiagen). The extracted DNA was completely sequenced by Macrogen (Seoul, Korea) using PacBio RS single-molecule real-time (SMRT) sequencing and Illumina HiseqXten sequencing. The PacBio SMRT reads were assembled using the hierarchical genome assembly process, and errors in PacBio sequencing were corrected by paired-end reads obtained by Illumina sequencing. The complete genome of the LAB isolate consisting of a chromosome and plasmid were deposited in GenBank under the accession numbers CP043431-2. Sequences were further annotated by RAST server using the classic scheme (https://rast.theseed.org/FIG/rast.cgi ). Protein coding sequences (CDSs) encoded on the plasmid were annotated by using NCBI BlastX (https://blast.ncbi.nlm.nih.gov/Blast.cgi ).
Hiseq x ten sequencing
Manufactured by Illumina
Sourced in United States
The HiSeq X Ten is a high-throughput DNA sequencing system designed for large-scale human genome sequencing. It employs Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data. The HiSeq X Ten can sequence up to 18,000 human genomes per year, making it a powerful tool for large-scale genomic research and clinical applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Truseq dna pcr free library preparation, by Illumina (2 mentions)
Truseq nano dna library preparation, by Illumina (2 mentions)
Hiseq x ten sequencer, by Illumina (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Minelute kit, by Qiagen (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Truseq dna library prep kit, by Illumina (1 mentions)
Nextseq 550, by Illumina (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Vahts stranded mrna seq library prep kit, by Vazyme (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Nanodrop spectrophotometer, by Agilent Technologies (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
15 protocols using hiseq x ten sequencing
1
Complete Genome Sequencing of LAB Isolate
2
Comprehensive Cancer Gene Profiling from FFPE Samples
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NGS analyses were performed in a centralized clinical testing centre according to protocols reviewed and approved by the Ethics Committee of Beijing Cancer Hospital. DNA was extracted from FFPE tumour tissues using a DNA Extraction Kit (QIAamp DNA FFPE Tissue Kit) according to the manufacturer’s protocols. Then, the DNA was sheared into 150–200 bp fragments with a BioruptorRPico Instrument (Diagenode, Seraing, Belgium). For each sample, 200–500 ng of FFPE DNA was then used for library preparation and quantification. Fragmented DNA libraries were constructed by the KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, Massachusetts, United States) following the manufacturer’s instructions. The final library was more than 600 ng, and fragment lengths were within the range of 250–400 base pairs (bp). DNA libraries were captured with a designed panel of 620 key cancer-related genes (GloriousMed, Shanghai, China). The list of 620 genes are shown in Supplementary Table S2 . The captured samples were subjected to Illumina HiSeq X-Ten sequencing with a minimum depth of 500 ×.
3
ATAC-seq of Early Zebrafish Embryos
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The ATAC-seq libraries of early zebrafish embryos were prepared as previously described with minor modifications (Wu et al. 2016 (link), 2018 (link)). In brief, embryos developing to desired stages were collected (20 embryos at the 256-cell stage, five embryos at the 1k-cell stage, and 12 embryos at the oblong stage) and then lysed in 200 µL of lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-40) for 10 min on ice to prepare the nuclei. After lysis, nuclei were collected by centrifugation at 500g for 5 min and then incubated with Tn5 transposase and tagmentation buffer (4 μL ddH2O, 4 μL 5 × TTBL, 5 μL TTE mix V5) at 37°C for 30 min (Vazyme TD502). After tagmentation, 5 μL of 5 × TS stop buffer were added and incubated at room temperature for 5 min to end the reaction. Fragmented DNA was then purified using a Qiagen MinElute kit (Qiagen 28004). PCR was performed to amplify the library by mixing 5 μL of N5XX primer, 5 μL of N7XX primer (Vazyme TD202) with 10 μL of 5 × TAB and 1 μL of TAE (Vazyme TD502) at the following PCR conditions: 72°C for 3 min; 98°C for 30 sec; 14 cycles at 98°C for 15 sec, 60°C for 30 sec and 72°C for 3 min; and 72°C for 5 min. After PCR, the libraries were purified with 1.4× Agencourt AMPure XP beads (Beckman Coulter Genomics, p/n A63881) and sequenced by the Illumina HiSeq X Ten sequencing platform.
4
Genome-wide DNA Methylation Analysis of Brain Tissue
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gDNA isolated from brain tissue samples was subjected to bisulfite conversion using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA, USA), and then a sequencing library was constructed using the Illumina TruSeq DNA Library Prep Kit (San Diego, CA, USA). Paired-end sequencing was performed on an Illumina HiSeq X Ten sequencing instrument, yielding 150-bp paired-end reads.
RNA sequencing libraries were generated from total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). Each library was diluted to 8 pM and subjected to 76 cycles of paired-read sequencing on an Illumina NextSeq 550 instrument.
RNA sequencing libraries were generated from total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). Each library was diluted to 8 pM and subjected to 76 cycles of paired-read sequencing on an Illumina NextSeq 550 instrument.
5
Transcriptomic Analysis of Triptolide Liver Effects
6
Developmental ATAC-seq of Zebrafish Embryos
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Embryos for ATAC-seq were harvested at five different developmental stages: 64-cell (2 hpf), 256-cell (2.5 hpf), 1k-cell (3 hpf), oblong (3.7 hpf), and dome (4.5 hpf). The ATAC-seq analyses of the zebrafish embryos involved a slightly modified approach that was based on the original method (Buenrostro et al. 2013 (link)); see Supplemental Methods . The libraries were sequenced by Illumina HiSeq X Ten sequencing. Public ATAC-seq data were collected from Bogdanovic et al. (2016) (link) (Supplemental Table S2 ).
Sequenced reads were mapped to the zebrafish genome (zv9 assembly) using Bowtie 2 (version 2.2.3) with default parameters (Langmead and Salzberg 2012 (link)), and peak calling was performed using MACS (version 1.4.2 20120305) (Zhang et al. 2008 (link)) with the following parameters: -f BED −g 1.4 × 109 --keep-dup all --nomodel --shiftsize 25. Peaks with P-value ≤ 1 × 10−10 and fold ≥10 were kept. SeeSupplemental Methods for details.
Sequenced reads were mapped to the zebrafish genome (zv9 assembly) using Bowtie 2 (version 2.2.3) with default parameters (Langmead and Salzberg 2012 (link)), and peak calling was performed using MACS (version 1.4.2 20120305) (Zhang et al. 2008 (link)) with the following parameters: -f BED −g 1.4 × 109 --keep-dup all --nomodel --shiftsize 25. Peaks with P-value ≤ 1 × 10−10 and fold ≥10 were kept. See
7
Illumina Sequencing and Variant Analysis of Tetraploid Cotton
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Genomic DNA from the parents used in this study, MC-WL, MC-BL, PD-3, and PD-93002, was isolated from ~100mg of fresh root radical tissue as previously described (Fang et al., 2010 (link)) and used for Illumina HiSeq X Ten Sequencing (Novogene Corp., Chula Vista, CA, USA) using 150bp paired-end sequencing runs. Equal amounts of the four indexed libraries were run together on three lanes of an Illumina flow cell. Sequence reads were aligned to the draft G. hirsutum TM-1 reference genome as well as a pseudo-tetraploid genome composed of the reference sequences from two diploids, Gossypium raimondii Ulbr., and G. arboreum (Paterson et al., 2012 (link); Li et al., 2014 (link); Zhang et al., 2015 ) with GSNAP software (Wu and Nacu, 2010 (link)). Polymorphisms were identified with bcftools software and by manual inspection of alignment files (Li et al., 2009 (link); Li, 2011 (link)). Select single nucleotide polymorphisms (SNPs) were converted to subgenome specific primers and scored on the F2 populations as before (Thyssen et al., 2014 (link)), which also allowed detection of the structural variation using primers flanking the boundaries of the 1.4Mb inversion (Supplementary Tables S1 and S2 at JXB online).
8
Genome Sequencing of Geosmithia purpuraceus
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Genome sequencing requires high-quality DNA [36 (link)]. The purity, integrity and concentration of the extracted DNA from G. purpuraceus were checked by Nanodrop, Qubit and 0.7% agarose gel electrophoresis. Genome survey sequences were obtained by the Illumina HiSeq X Ten sequencing platform, which can provide rapid and accurate basial information such as the genome size, heterozygosity and repeat content by the k-mer method [37 (link)]. Using the software “kmer_freq_stat” independently developed by Beijing Biomarker Technologies, the heterozygosity rate of G. purpuraceus genome was estimated to be 0.01% (Figure S1 ), which met the requirements for whole-genome construction. Then, the whole genome was sequenced by the Oxford Nanopore sequencing platform. About 15.13 Gb of raw data were generated, and the quality control of data was performed with the sequence length ≥ 2000 bp, which was subsequently corrected by Canu v1.5 [38 (link)]. Then, the corrected reads were used for genome assembly and adjusted with wtdbg2 and Racon [39 (link)]. Finally, the highly accurate genome was achieved by the correction and optimization performed with Pilon v1.14 combined with the second-generation sequencing data [40 (link)]. Genome assembly integrity was assessed through BWA and BUSCO v4.1.2 software [41 (link),42 (link)].
9
Illumina RNA-Seq Library Preparation
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RNA samples from Ou, Os, Mz4, and Gc samples were used for Illumina (San Diego, CA, USA) library construction and sequencing, respectively. The cDNA libraries for Illumina HiSeq X Ten sequencing were constructed as follows: mRNA was enriched from the total RNA using the magnetic oligo-dT bead binding method and sheared into short fragments using fragmentation buffer. Then, the short mRNAs were used as templates to synthesize double-stranded cDNAs using random primers by reverse transcription. The cDNA fragments were purified using a QiaQuick PCR extraction kit (Qiagen, Venlo, Netherlands) and ligated with Illumina sequencing adapters. The ligation products were size-selected by agarose gel electrophoresis and enriched by PCR to construct the cDNA libraries, which were sequenced on the Illumina HiSeq X Ten platform. All the sequencing works were carried out at Wuhan Frasergen Bioinformatics Co., Ltd. (Wuhan, China). After RNA sequencing (RNA-Seq), the raw reads were filtered by removing adaptors, reads containing more than 10% of unknown nucleotides, and low-quality reads. The Q20/Q30 and GC content of the clean reads were calculated.
10
DNA Extraction and Whole-Genome Sequencing Protocol
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