The biopsied mucosal samples were transferred to Majorbio (Shanghai, China) where the total DNA was extracted, amplified and sequenced according to their standardized protocol19 (link). The V3- >V1 region of the 16S ribosomal subunit gene was amplified using 27F/533R barcoded primers and sequenced using a Roche Genome Sequencer FLX+). The sequencing results were archived in the Short Reads Archive (PRJNA285379).
Genome sequencer flx
Manufactured by Roche
Sourced in United States
The Genome Sequencer FLX is a lab equipment product designed for high-throughput DNA sequencing. It utilizes a pyrosequencing-based technology to rapidly generate sequence data from DNA samples. The core function of the Genome Sequencer FLX is to perform efficient and accurate DNA sequencing to support various applications in genomic research and analysis.
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Lab products found in correlation
Dneasy blood tissue kit, by Qiagen (2 mentions)
Titanium reagents, by Roche (1 mentions)
Biorobot ez1, by Qiagen (1 mentions)
Paxgene blood rna tube, by Preanalytix (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
Allprep mini kit, by Qiagen (1 mentions)
Gs de novo assembler, by Roche (1 mentions)
Gsassembler, by Roche (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
T3000 thermocycler, by Analytik Jena (1 mentions)
Superscript 3 reverse transcriptase and oligo dt primers, by Thermo Fisher Scientific (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Newbler 2, by Roche (1 mentions)
100 bp ladder marker, by Thermo Fisher Scientific (1 mentions)
Genome analyzer iix, by Illumina (1 mentions)
Gs flx titanium rapid library preparation kit, by Roche (1 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (1 mentions)
28 protocols using genome sequencer flx
1
16S rRNA Amplicon Sequencing Protocol
2
Genomic DNA sequence analysis of strain W5052
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The genomic DNA of the strain W5052 was purified from the BHI culture medium using a DNeasy Blood & Tissue Kit (Qiagen). The sequence of the genomic DNA was determined by a Roche Genome Sequencer FLX. This analysis was carried out by Hokkaido System Science Inc. (Hokkaido, Japan). Jemboss, a computer software program, was used to predict possible open reading frames [23 (link)]. Homologues to the deduced amino acid sequence were searched using the BLAST program [24 (link)]. A multiple sequence alignment was performed using the Genetyx software program (Software Development Inc., Tokyo, Japan).
3
Microbial Community Profiling via NGS
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Next-generation DNA sequencing can provide in-depth descriptions of microbial communities. Samples were prepared to analyze the microbial community structures based on next-generation sequencing of the V3 region in samples collected from upper site 1 and lower site 4 of the sampling locations A and E, respectively. The following pair of primers was used to amplify the hypervariable V3 region (approximately 500 bp): 27F-5′-AGAGTTTGATCCTGGCTCAG-3′ and 533r-5′-TTACCGCGGCTGCTGGCAC-3′. The samples were individually barcoded to enable multiplex sequencing. PCR reactions were conducted in a 20-μl volume: 4 μl of 5× FastPfu Buffer; 2 μl of dNTPs (2.5 mM); 0.4 μl of forward primer (5 μM), 0.4 μl of reverse primer (5 μM), 0.4 μl of Fastpfu polymerase and 10 ng of template DNA. The following amplification protocol was used: 5 min initial denaturation at 94°C; 11 cycles of denaturation at 95°C, annealing at 60°C for 30 s, and extension at 72°C for 1 min; 25 cycles of denaturation at 95°C, annealing at 50°C for 30 s, and extension at 72°C for 1 min, and finally extension at 72°C for 10 min. DNA molecules marked with different barcodes were sequenced using a Roche Genome Sequencer FLX+ (Roche 454 Life Sciences, Branford, CT, USA) at Shanghai Majorbio Bio-pharm Technology, Co., Ltd.
4
BCR Repertoire Analysis by NGS
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The linear amplification protocol has been extensively described earlier.14 Samples were prepared for sequencing according to the manual for amplicon sequencing and sequenced on a Roche Genome Sequencer FLX (Titanium platform; Roche Diagnostics, Almere, The Netherlands). For each sample 10,000 BCRheavy sequences were analyzed. NGS visualizes expanded BCR clones as a deviation in the repertoire because the BCR sequence they carry is determined more frequently than low abundant BCR sequences and thus account for a relatively large proportion of all the sequences found. Moreover, it is not unlikely that plasmacytoid cells are identified in peripheral blood; these cells produce increased amounts of BCR messenger RNA, producing a comparable deviation in the repertoire as expanded B cells. For clarity, we will use the term “dominant clone” to denote these clones.
5
Pyrosequencing-based Microbial Community Analysis
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Pyrosequencing was performed on a Roche Genome Sequencer FLX+ using Titanium chemistry by Macrogen (Roche Applied Science, Mannheim, Germany). Three standard flow-gram format (SFF) files were generated by 454 pyrosequencing. The SFF file was analyzed by the software package mothur (version 1.33.2) following the protocol provided by https://mothur.org/wiki/454_SOP. Briefly, De-noising and chimera analysis conducted with the AmpliconNoise (Quince et al., 2011) and UCHIME algorithms were used to reduce sequence errors. Furthermore, quality trimming was conducted to remove unwanted sequences shorter than 200 bp and reads containing ambiguous bases and with homopolymers longer than 8 bases.
Remaining sequences were used to identify unique sequences by aligning with the SILVA-based bacteria reference alignment. Within unique sequences, the Uchime tool was applied to remove chimeras. Next, "Chloroplast", "Mitochondria", or "unknown" were identified and removed from the dataset. Subsequently, after calculating the pairwise distance and generating the distance matrix, a 97% identity threshold was used to cluster sequences into Operational Taxonomic Units (OTUs) according to the UCLUST algorithm (Edgar, 2010) . For each OTU, the SILVA database was applied to annotate taxonomic information.
Remaining sequences were used to identify unique sequences by aligning with the SILVA-based bacteria reference alignment. Within unique sequences, the Uchime tool was applied to remove chimeras. Next, "Chloroplast", "Mitochondria", or "unknown" were identified and removed from the dataset. Subsequently, after calculating the pairwise distance and generating the distance matrix, a 97% identity threshold was used to cluster sequences into Operational Taxonomic Units (OTUs) according to the UCLUST algorithm (Edgar, 2010) . For each OTU, the SILVA database was applied to annotate taxonomic information.
6
454 GS FLX Titanium Mate Pair Sequencing
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3-kb and 8-kb mate pair libraries were constructed according to the 454 GS FLX Titanium paired-end protocol with Titanium reagents (Roche) and sequenced on a full PicoTiterPlate on a Genome Sequencer FLX+ Instrument. Sequencing data were processed and bases called using the Roche 454 Software Version 2.6 (shotgun sequencing data processing pipeline).
7
NFkB1 Gene Sequencing from Whole Blood
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Genomic DNA was isolated from the collected whole blood samples that were stored in PAXgene Blood RNA tubes (PreAnalytix) using a QIAmp DNA Mini Kit and the EZ1 BioRobot (QIAgen Inc., Mississauga, ON, Canada). The genomic regions of NFκB1 were amplified from the genomic DNAs by overlapping PCRs with the 14 gene-specific pair primers (Supplementary Table S1 ). The amplified PCR products were confirmed by agarose gel electrophoresis and purified, quantified, and tagged with sequence tags. Sequencing was performed on Genome sequencer FLX from 454 Life Sciences (Roche 454) at Genomic Core, National Microbiology Laboratory (NML). All the sequencing data was submitted to the NCBI BioProject database (Bioproject ID: PRJNA752377).
8
Amplification and Sequencing of IGHV Genes
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Linear amplification of the IGHV genes was based on the protocol used previously (29 (link), 30 (link)) using primer sets (available upon request) that covered all functional IGHV genes. Briefly, the IGHV region primers contained a primer B sequence required for amplicon sequencing (Roche Diagnostics, Mannheim, Germany). Amplified products were purified and used in a generic PCR using primer B as forward primer and a reverse generic primer specific for all functional IGHJ genes, containing primer sequence A.
Samples were again purified, quantified, prepared for sequencing according to the manual for amplicon sequencing, and sequenced on a Roche Genome Sequencer FLX (titanium platform).
The bioinformatics pipeline used to obtain the IGHV sequences was done by performing Multiplex Identifier sorting, identification of gene segments, and removal of artifacts.
Samples were again purified, quantified, prepared for sequencing according to the manual for amplicon sequencing, and sequenced on a Roche Genome Sequencer FLX (titanium platform).
The bioinformatics pipeline used to obtain the IGHV sequences was done by performing Multiplex Identifier sorting, identification of gene segments, and removal of artifacts.
9
Profiling Staphylococcus Populations via 16S Sequencing
10
Gut Microbiome Profiling by 16S rRNA Sequencing
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For 16S rRNA gene-based amplicon sequencing analysis, DNA from individual faecal samples (n 8) before the start of the study (day 1) and caecal samples (n 8) at the end of the study was extracted using the FastDNA SPIN Kit for soil. Extracted DNA was sent to DNAVision for high-throughput sequencing of the hypervariable V5-V6 region of the entire 16S rRNA gene pool using a 454 Life Sciences Genome Sequencer FLX instrument (Roche AG), according to procedures described previously (29) . Resulting sequencing reads were quality-filtered according to the following three criteria: maximum of one mismatch in barcode and primers, at least 240 nucleotides length and a maximum of two undetermined bases per sequence. Sequencing reads passing the quality check were assigned at the phylum, family and genus level using the Ribosomal Database Project (RDP) Bayesian classifier (v2.1) (30) with a confidence threshold of 80 %. Sequences were further assigned into operational taxonomic units (OTU) based on nearest neighbour clustering using mothur software package (31) . Chao1 richness and Shannon diversity were calculated based on the numbers of OTU. The complete 16S rRNA gene-based amplicon sequencing data set has been deposited to the National Center for Biotechnology Information Sequence Read Archive under accession number SRP044704.
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