A total of 454 sequencing libraries were generated from the total RNA samples of the early and full flowering stages of Carthamus tinctorius using a GS-FLX sequencer (Roche, Basel, Switzerland). Clean reads were obtained, were followed by the deletion the raw data including low-quality reads, adapter reads, hairpin structural reads and shorter reads (<50 bp), and were then assembled into unigenes using the MIRA program [20 (link)]. The longest transcripts were further selected as unigenes for functional analysis by identifying their corresponding nucleotide sequences. The study of functional annotations and pathway enrichment analysis of the unigenes were investigated using a non-redundant protein sequence (http://www.ncbi.nlm.nih.gov ) and non-redundant nucleotide sequences (http://www.ncbi.nlm.nih.gov/ ). Further annotations were analyzed in SwissProt. Gene ontology (GO) annotations were carried out using blast2GO (http://www.geneontology.org/ ) [21 (link)]. To determine the high-level functional enrichment of biosynthetic systems from molecular-level information, the Kyoto Encyclopedia of Genes and Genomes pathways analysis was performed using GenMAPP 2.1 (http://www.genmapp.org/ ).
Gs flx sequencer
Manufactured by Roche
Sourced in Spain
The GS-FLX sequencer is a next-generation DNA sequencing platform developed by Roche. It utilizes pyrosequencing technology to determine the nucleotide sequence of DNA samples. The core function of the GS-FLX sequencer is to generate high-throughput, high-quality DNA sequence data for various applications in genomics, genetics, and molecular biology research.
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Lab products found in correlation
Picotiterplate, by Roche (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Modulus 9200 fluorimeter, by Promega (1 mentions)
Nucleo fast pcr purification kit, by Macherey-Nagel (1 mentions)
Ampure xp beads, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Gs flx titanium sequencing kit, by Roche (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Nucleospin soil kit, by Macherey-Nagel (1 mentions)
Abi prism 377, by Thermo Fisher Scientific (1 mentions)
Gene clean turbo, by Qbiogene (1 mentions)
Newbler software, by Roche (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
12 protocols using gs flx sequencer
1
Transcriptome Analysis of Carthamus tinctorius Flowering
2
Fecal Metagenome DNA Extraction and Sequencing
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The fecal samples were resuspended in sterile PBS [containing, per liter, 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 (pH 7.2)] and centrifuged at 1250 g and 4°C for 2 min to remove fecal debris. The supernatants were centrifuged at maximum speed at 4°C for 5 min to pellet the cells. DNA was extracted with the QIAamp® DNA Stool Kit (Quiagen) following the manufacturer's instructions. Total DNA integrity was checked by running a standard agarose gel electrophoresis and the concentration was quantified with the QuantiT PicoGreen dsDNA Assay Kit (Invitrogen). For each sample, except of F_after from which there was no enough amount of DNA, the total DNA (metagenome) was directly pyrosequenced with a Roche GS FLX sequencer and Titanium chemistry in the Center for Public Health Research (FISABIO-Salud Pública) (Valencia, Spain). Thus, a total of 12 metagenomes were analyzed. We obtained a mean of 78,976 reads per sample with an average length of 374 bp.
3
DNA Library Sequencing Quality Metrics
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The enriched, clonally amplified DNA library was combined with the required XLTF Control Article (control beads) and sequenced on two full sequencing plates (PicoTiterPlate [PTP]; Roche) on the GS FLX sequencer following current Roche sequencing procedures. The total keypass (an assessment of the efficiency of loading the sample onto the PTP), total filterpass (an assessment of the overall DNA sequence [“read”] quality), and average read length value for the DNA library was reported in the corresponding 454QualityFilterMetrics.txt and 454Base CallerMetrics.txt files at the completion of the sequencing run. With the exception of the average read length, these values were assessed against defined acceptance criteria. There was no acceptance criterion for average read length due to the variability and nature of the input sample types.
4
High-Throughput Sequencing and Bioinformatic Analysis
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Prior to sequencing, the quality and quantity assessment of amplicons was done using the Bioanalyzer (Agilent, Santa Clara, CA, USA) and fluorometer by Inqaba Biotec, Pretoria, South Africa. Approximately 2 μg (at a concentration of at least 50 ng/μL) of purified DNA samples were sequenced on a GS-FLX sequencer using the 454 high-throughput pyro-sequencing technology (Roche Diagnostics, Mannheim, Germany) by Inqaba Biotec, Pretoria, South Africa. After sequencing, the data from the 454-read sequences of each sample were assembled into contigs using the proprietary Roche 454 Newbler Assembler software. Not all reads were assembled into contigs for each sample set and these are indicated as singletons. Subsequently, the sequences were annotated using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov ). Similarities at the nucleotide level were identified using BLASTN and protein homologies were identified using the non-redundant protein databases BLASTX [9 (link)]. Each gene was then placed into a functional category based on the putative function thereof.
5
454 Sequencing of Normalized cDNA Library
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The normalized cDNA library was sequenced using a 454 Life Sciences GS-FLX sequencer (Roche / 454 Life Sciences, Branford, CT, USA) utilizing a protocol to resolve 400 base-pair (bp) reads. Briefly, cDNAs were nebulized and size-selected for 500 to 800 bp fragments. Two primer sequences, Adaptor A and Adaptor B, were ligated to the fragments. cDNAs containing both an A and a B adaptor were melted into single stranded DNA, immobilized onto DNA capture beads and emulsified in oil for polymerase chain reaction (emPCR). The emPCR was titrated to determine the optimal amount of single stranded DNA (ssDNA) needed to create a 1:1 DNA fragment to bead ratio. emPCR was performed and the amplified library was loaded onto a 70 × 75 mm PicoTiterPlate (Roche / 454 Life Sciences) and sequenced for one full plate run.
6
16S rRNA Gene Amplification and Pyrosequencing
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Single-stranded cDNA was constructed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Grand Island, NY) in 20 µl reactions, with several modifications as previously described5 (link). Universal bacterial primers 8 F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 533 R (5′-GCCTTGCCAGCCCGCTCAGGC-3′) were used to partly amplify the 16S rRNA gene from the single-stranded cDNA in two 50 µl reactions, following the PCR and purification conditions previously described20 (link). The 500-bp PCR products were purified with the Nucleofast PCR purification kit (Macherey-Nagel, Düren, Germany) and further cleaned by AMPure XP beads (Roche, Basel, Switzerland) before pyrosequencing. The final DNA per sample was measured by fluorescence with the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific) in a Modulus 9200 fluorimeter (Turner Biosystems, Sunnyvale, CA, USA) so samples could be mixed in equimolar amounts. PCR products were pyrosequenced from the forward primer end only by using a GS-FLX sequencer with Titanium chemistry (Roche). One-eighth of a plate was used for each pool of 20 samples, which were amplified with a different forward primer containing a unique 8-bp “barcode.”
7
Amplicon Library Sequencing Protocol
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Sequencing was performed by the NIH Intramural Sequencing Center (NISC). For each sample, the amplicon library concentrations were determined with quantitative PCR (qPCR) using Library Quantification Kit −454 Titanium (Lib-L)/Universal (Kapa Biosystems) on a CFX96 Real-Time System (Bio-Rad). The libraries were then amplified on Capture Beads by emulsion PCR using a ratio of DNA molecules: DNA Capture Bead between 0.5 and 2.0 and GS Titanium emPCR Kit (Lib-L) and emPCR Breaking Kits (Roche) following the manufacturer's instructions. Approximately 0.7–1.0 × 106 enriched Capture Beads were applied to each region of a two-region PicoTiterPlate (70 × 75) and sequenced on a GS FLX Sequencer (Roche-454 Life Sciences, Bradford, CT) using GS FLX Titanium Sequencing Kit for 200 cycles. Post-run signal processing, quality filtering (modified according to recommendations in Roche-454 Life Sciences Application Brief No. 001-2010) and conversion of flowgram intensities to nucleotide sequence and Phred-based quality scores were performed on an off-instrument linux cluster running 454 application software version 2.6.
8
Colonic Crypt and Goblet Cell Analysis
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Transverse sections of the proximal colon were collected and stored in 10% formalin solution at room temperature for subsequent assessment of colonic crypt length and goblet cell number.
In preparation for microbial analysis, approximately 50 mg of colon digesta DNA was extracted using a NucleoSpin Soil kit accordingly to the manufacturer's instructions (Macherey Nagel, Düren, Germany). For pyrotag sequencing, the V4‐V6 region of the bacterial 16S rRNA gene was amplified. The primers and PCR conditions used have been previously published by our group25 . Purified products were pooled in equivalent quantities and sent to Macrogen (Seoul, Korea) for unidirectional sequencing from the forward primer using the Roche GS‐FLX sequencer with Titanium chemistry. Sequences were analyzed accordingly with a method previously described 25 .
In preparation for microbial analysis, approximately 50 mg of colon digesta DNA was extracted using a NucleoSpin Soil kit accordingly to the manufacturer's instructions (Macherey Nagel, Düren, Germany). For pyrotag sequencing, the V4‐V6 region of the bacterial 16S rRNA gene was amplified. The primers and PCR conditions used have been previously published by our group
9
Recombinant P. eryngii AAO Variants
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Native AAO from P. eryngii was obtained by expression in E. coli of the mature AAO cDNA (GenBank AF064069), followed by in vitro activation in the presence of the FAD cofactor, and purification by ion-exchange chromatography as described previously [45] . Mutated variants were prepared using the QuikChange site-directed mutagenesis kit (Stratagene). For the PCR reactions, the cDNA cloned into the pFLAG1 vector was used as template, and the following oligonucleotides (direct sequences) bearing mutations (italics) as primers: i) Y92L, 5'-GGG TCT AGC TCT GTT CAC CTC ATG GTC ATG ATG CG-3'; ii) Y92F, 5'-GGG TCT AGC TCT GTT CAC TTC ATG GTC ATG ATG CG-3';and iii) Y92W, 5'-GGG TCT AGC TCT GTT CAC TGG ATG GTC ATG ATG CG-3'. Mutations were confirmed by sequencing (GS-FLX sequencer from Roche) and the mutated variants were obtained as described for recombinant AAO. Naturally-oxidized AAO concentration was determined using the molar absorbances of native AAO and of its Y92L, Y92F and Y92W variants ( 463 11050 M -1 cm -1 , 463 11240 M -1 cm -1 , 463 10044 M -1 cm -1 , 457 10693 M -1 cm -1 , respectively) calculated by heat denaturation and estimation of the free FAD released ( 450 11300 M -1 •cm -1 ) [45] .
10
Genome Sequencing and Assembly of Azoarcus sp. CIB
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Genome sequence, contigs assembling, and gaps filling Azoarcus sp. CIB was anaerobically grown at 30 ºC in MC medium [32] containing 3 mM benzoate as sole carbon and energy source and 10 mM nitrate as electron acceptor. Cultures were collected when they reached the early stationary phase and genomic DNA was extracted using previously published protocols [32] .
The genome sequencing of Azoarcus sp. CIB was carried out using the 454 Life Sciences high-density pyrosequencing methodology in a GSFLX sequencer from Roche at LifeSequencing (Valencia, Spain). FASTQ reads (about 250-nt long) were assembled in contigs by using the Newbler software from Roche. Contigs were ordered in scaffolds by performing a long-tag paired-end sequencing according to Roche protocols at LifeSequencing (Valencia, Spain). Gap filling on the scaffolds was performed by manual assembly of FASTQ reads with BioEdit (Ibis Biosciences) and by conventional sequencing methods (ABI Prism 377; Applied Biosystems) of PCR products (purified with Gene Clean Turbo, Q-BIOgene) spanning the regions between flanking contigs.
The genome sequencing of Azoarcus sp. CIB was carried out using the 454 Life Sciences high-density pyrosequencing methodology in a GSFLX sequencer from Roche at LifeSequencing (Valencia, Spain). FASTQ reads (about 250-nt long) were assembled in contigs by using the Newbler software from Roche. Contigs were ordered in scaffolds by performing a long-tag paired-end sequencing according to Roche protocols at LifeSequencing (Valencia, Spain). Gap filling on the scaffolds was performed by manual assembly of FASTQ reads with BioEdit (Ibis Biosciences) and by conventional sequencing methods (ABI Prism 377; Applied Biosystems) of PCR products (purified with Gene Clean Turbo, Q-BIOgene) spanning the regions between flanking contigs.
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