The genomic DNA was extracted from peripheral blood leukocytes using the QIAGEN MagAttract HMW DNA Kit (QIAGEN, Germany) following the manufacturer’s protocol. An stLFR library was constructed using the MGIEasy stLFR Library Prep Kit (MGI Tech, China) according to the manufacturer’s instructions. Briefly, transposons were inserted into long DNA molecules. Subsequently, these transposon-inserted DNA sequences were hybridized with clonal barcoded beads and then ligated with barcoded oligo and adapters via splint oligo. After adding the library adapters, PCR amplification and circularization were performed to generate DNA nanoballs (DNBs). The prepared library was then sequenced on the MGISEQ-2000 platform (MGI Tech) with a 100 bp paired-end strategy. The expected raw data of each sample is 100GB or more.
Mgiseq 2000 platform
Manufactured by MGI Tech
Sourced in China
The MGISEQ-2000 platform is a high-throughput DNA sequencing system developed by MGI Tech. It utilizes a proprietary sequencing technology to perform parallel sequencing of DNA samples. The core function of the MGISEQ-2000 platform is to generate high-quality sequencing data for various applications in the life sciences and biomedical research fields.
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Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Rna 6000 pico kit, by Agilent Technologies (2 mentions)
Mgieasy rna directional library prep set, by MGI Tech (2 mentions)
Fcl pe100, by MGI Tech (2 mentions)
Magattract hmw dna kit, by Qiagen (1 mentions)
Mgieasy stlfr library prep kit, by MGI Tech (1 mentions)
Qiaamp viral rna, by Qiagen (1 mentions)
Qubit rna br assay kit, by Thermo Fisher Scientific (1 mentions)
Dnbseq g400, by MGI Tech (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Repli g single cell kit, by Qiagen (1 mentions)
Dna free dna removal kit, by Thermo Fisher Scientific (1 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyze, by Agilent Technologies (1 mentions)
Vahts universal v6 rna seq library kit for mgi, by Vazyme (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
13 protocols using mgiseq 2000 platform
1
Long-Read Genomic DNA Extraction and stLFR Library Prep
2
SARS-CoV-2 Genome Sequencing and Phylogenetic Analysis
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Total viral RNA from TA was extracted and quantified with the QIAamp Viral RNA (Qiagen, Germany) and the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, CA, USA), respectively. cDNA libraries were constructed with the ATOPLex SARS-CoV-2 full-length genome panel v1.0 (kindly donated by MGI Tech Co., Shenzhen, China), an amplicon-based strategy to improve sequencing readout. Dual-indexed, single-stranded library pools were converted to DNA nanoballs by rolling circle amplification and submitted to pair-end sequencing (100 nt) on the MGISEQ-2000 platform (recently named DNBSEQ-G400, MGI Tech Co. Ltd., Shenzhen, China).
Genomic sequences were quality scored, filtered, trimmed, and assembled into contigs using Genome Detective (https://www.genomedetective.com/ ) [39 (link)]. Consensus fasta sequences were aligned with ClustalW in Unipro UGENE [40 (link)] (version 38), and phylogenies were constructed with Nextclade [41 (link)] to assign the emerging clades (Supplementary Table 2 ).
Genomic sequences were quality scored, filtered, trimmed, and assembled into contigs using Genome Detective (
3
Plasma ctDNA Evaluation by Signatera Assay
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Plasma ctDNA was evaluated by the Huajianwei bespoke MRD assay based on Signatera as previously described13 (link),31 (link). Briefly, 16 top-ranked SNVs were selected based on the tumor tissue whole exome sequencing (WES) data generated from a pan-cancer WES panel (Quanxi) on the MGISEQ-2000 platform (MGI Tech), and 16-plex specific primer pairs were used to amplify the universal cfDNA libraries. The products were then sequenced on the HiSeq2500 system (Illumina Inc). Detailed methods are described in the Supplemental Digital Content 2, http://links.lww.com/JS9/C41 . To minimize the false positive rate, the plasma sample with at least two variants positive (out of 16 variants in total per person) was defined as ctDNA or MRD positive, and ctDNA was quantified in mean tumor molecule per milliliter (MTM/ml) plasma (Supplementary eTable 3, Supplemental Digital Content 3, http://links.lww.com/JS9/C42 ). The raw data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA)32 of China National GeneBank DataBase (CNGBdb)33 (link) with accession number CNP0005016 (https://db.cngb.org/ ).
4
Maize and Rice F1 Hybrid Hi-C
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The maize F1 hybrid (B73 × SK) seeds were from our own lab in Huazhong Agricultural University. The rice F1 hybrid (MH63 × ZS97) seeds were provided by Dr. Xiangchun Zhou in Huazhong Agricultural University. The maize F1 hybrids were planted in 2020 Wuhan, China. The rice F1 hybrids were planted in 2021 Wuhan, China. Young root tissues of F1 hybrid were harvested. Hi-C proximity libraries were constructed by the previously described method with restriction enzyme MboI [4 (link)]. The libraries were size-selected to retain 350 bp DNA fragments and sequenced on MGISEQ2000 platform (MGI-Tech) to generate 150 bp paired-end reads.
5
Single-Cell DNA Sequencing of Gametes
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The seeds of F1 hybrid were planted and then immature tassels were harvested before they had emerged. Gamete cells (microspores) were isolated from tetrads as described in a previous study [23 (link)]. DNA was extracted from each gamete cell using QIAGEN REPLI-g Single Cell Kit (Cat No. 150343), followed by multiple displacement amplification (MDA) [22 (link)] procedure to generate enough DNA for downstream experiments. A sequencing library was constructed for each gamete cell and then to be sequenced on MGISEQ2000 platform (MGI-Tech) to generate 150 bp paired-end reads.
6
Transcriptomic Analysis of Pepper Fruit Exocarp
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RNA samples taken from the exocarp of Chen12-4 and Chen12-4-VIGS690 (TRV2-CA10g11690 silencing lines) at 15 days post-anthesis (DPA) were analyzed using RNA-seq. The RNA samples were sent to Frasergen Bioinformatics Co., Ltd. to generate libraries and then sequence using an MGISEQ-2000 platform (MGI Tech Co. Ltd.). After filtration by BBTools (https://jgi.doe.gov/data-and-tools/bbtools ) to remove the low-quality reads, the clean reads were then aligned to the C. annuum CM334 genome (version 1.55) using the HISAT2 program (https://github.com/DaehwanKimLab/hisat2 ). The level of expression of mRNA was calculated by fragments per kilobase of transcript per million mapped reads (FPKM). DESeq software was used to standardize the number of mRNA counts in each sample. Finally, differentially expressed genes was detected according to the screening criteria of |log2 (foldchange)| ≥1 and FDR (False Discovery Rate) < 0.01.
7
Ovarian Total RNA Sequencing
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Total RNA samples were isolated from ovaries using TRIzol reagent (Invitrogen) according to the standard protocol and treated with DNase using the DNA-free DNA Removal Kit (Invitrogen). rRNA was removed by hybridization of 200 ng of total RNA with biotinylated oligonucleotides complementary to different regions of rRNA followed by binding to Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen). After precipitation, supernatants containing rRNA-depleted RNA were precipitated with isopropanol. NGS libraries were prepared in duplicates using MGIEasy RNA Library Prep Set V3.1 (MGI Tech Co. Ltd.) according to the manufacturer’s recommendations. Sequencing was carried out on the basis of the Center for Precision Genome Editing and Genetic Technologies for Biomedicine of the Pirogov Russian National Research Medical University (Moscow) with 2 × 100 bp paired-end reads on a MGISEQ-2000 platform (MGI Tech Co. Ltd.). RNA-seq data were deposited in the NCBI GEO under the accession number GSE183035.
8
Transcriptome Profiling via NGS
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RNA extracted from samples of total cellular lysate and polysome gradient fractions were quality-checked with the Bioanalyzer 2100, using the RNA6000Pico kit (Agilent Technologies, Santa Clara, CA, USA), as described above. DNA libraries were prepared using the MGIEasy RNA Directional Library Prep Set (MGI Tech, Shenzhen, China) according to the manufacturer’s instructions, and subjected to the next-generation sequencing (NGS) on the MGIseq-2000 platform, utilizing the 2x100 PE sequencing mode (FCL PE100, MGI Tech). All relevant procedures were performed in the SB RAS Genomics Core Facility (ICBFM SB RAS, Novosibirsk, Russia).
9
Metagenomic Library Construction and Sequencing
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DNA libraries were constructed using DNA fragmentation, end repair, adapter ligation, and PCR amplification by MetaCAP Pathogen Capture Metagenomic Assay Kit (KingCreate, Guangzhou, China). Agilent 2100 (Agilent, Santa Clara, CA, USA) was used for quality control of the DNA libraries. Quality-qualified libraries were pooled. Then, a DNA nanoball (DNB) was prepared and sequenced using the MGISEQ-2000 platform (MGI Tech, Shenzhen, China) set to 50-bp single end with average of 100 million reads per sample (15 (link)).
10
Whole Genome Sequencing with MGIEasy
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DNA extraction was performed using a magnetic bead extraction kit (MGIEasy, 1000006988, MGI Tech Co., Ltd., Shenzhen, China). A Qubit 3.0 Fluorometer (Q33216, Thermo Fisher Scientific, Waltham, MA, USA) was used for nucleic acid quantification. An MGIEasy FS DNA Library Prep Kit (MGIEasy, V1.0, 1000006988, MGI Tech Co., Ltd.) was used to construct the library. Agilent 2100 Bioanalyze (G2939AA, Agilent Technologies, Santa Clara, CA, USA) was used to detect the size of DNA fragments, and Qubit 3.0 was used to quantify the library. The WGS was performed on the MGISEQ-2000 platform (paired-ends, 100 bp; MGI Tech Co., Ltd.), with an average sequencing depth of 112×.
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