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454 pyrosequencing

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The 454 pyrosequencing is a DNA sequencing technology that utilizes sequencing-by-synthesis principles. It enables rapid, parallel, and high-throughput DNA sequencing. The system generates DNA sequence data by detecting the light signal produced during the incorporation of fluorescence-labeled nucleotides.

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Lab products found in correlation

454 gs flx system, by Roche (2 mentions) Large construction kit, by Qiagen (1 mentions) Ampure xp bead, by Beckman Coulter (1 mentions) Dnazol kit, by Thermo Fisher Scientific (1 mentions) Titanium chemistry, by Roche (1 mentions) Miseq instrument, by Illumina (1 mentions) Miseq platform, by Roche (1 mentions) 454 gs flx platform, by Roche (1 mentions) Miseq platform, by Illumina (1 mentions)

11 protocols using 454 pyrosequencing

1

MHC Class II Genotyping in Lemurs

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PCR amplification targeting the two loci of the most variable parts of the MHC class II region, DRB and DQB, was performed using primers that flank the functionally important ABS and captured the full variability in the congener M. murinus (Schad et al. 2004 ; Averdam et al. 2011 (link)). PCR reaction mix and amplification conditions are summarised in ESM 2. Each individual PCR product (further referred to as amplicon) was electrophoresed on 1 % agarose gel to verify successful amplification. Primer design and the preparation of locus-specific amplicon libraries were described elsewhere (Huchard et al. 2012 (link)). Sequencing was conducted according to standard protocols for GS Junior sequencing (Roche, 454 pyrosequencing). All sequencing reads retrieved from a total of six sequencing runs were processed according to a post-sequencing quality control procedure following Huchard et al. (2012 (link)).
Pechouskova E., Dammhahn M., Brameier M., Fichtel C., Kappeler P.M, & Huchard E. (2015). MHC class II variation in a rare and ecological specialist mouse lemur reveals lower allelic richness and contrasting selection patterns compared to a generalist and widespread sympatric congener. Immunogenetics, 67(4), 229-245.
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2

Metagenome-derived Enzyme Discovery Pipeline

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Based on functional screening, a total of 173 positive fosmid clones were randomly selected from variant levels of halo against screening substrates for full sequencing (Figure S1), including 68 endoglucanase-, 15 exoglucanase-, 40 β-glucosidase-, and 50 endoxylanase-positive clones (10 out of these clones exhibited dual enzyme specificities). After cell density normalization, every 10 to 12 different fosmids were pooled as a sample for the whole fosmid DNA extraction using the QIAGEN Large-Construction kit according to manufacturer’s instructions. Extracted DNA was directly subjected to 454 pyrosequencing (Roche Diagnostics, USA). After assembly (Newbler 2.5.3), coding regions and bacterial operons were predicted by FGENESB (http://linux1.softberry.com/berry.phtml). The predicted amino acid sequences were aligned to the eggNOG v4.5.1 database [35 (link)] and KEGG for further functional prediction (E-value <1.0e−5). Searches for carbohydrate-active enzymes was performed against dbCAN database with default parameters. Signal peptides were predicted by SignalP 4.1 in CBS (http://www.cbs.dtu.dk/services/SignalP/). Molecular masses and isoelectric points were predicted by ExPASy (http://www.expasy.ch/tools/protparam.html). Protein cellular location was predicted using CELLO v.2.5 (http://cello.life.nctu.edu.tw) [36 (link)].
Liu N., Li H., Chevrette M.G., Zhang L., Cao L., Zhou H., Zhou X., Zhou Z., Pope P.B., Currie C.R., Huang Y, & Wang Q. (2018). Functional metagenomics reveals abundant polysaccharide-degrading gene clusters and cellobiose utilization pathways within gut microbiota of a wood-feeding higher termite. The ISME Journal, 13(1), 104-117.
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3

Targeted Sequencing of Somatic Variants

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Primers were designed to amplify 300-500bp fragments by conventional PCR for putative single nucleotide substitutions identified by exome sequencing. PCR amplification was performed for both tumour and remission DNA pairs and fragments were purified using SPRI bead clean up (Agencourt AMPure XP beads, Beckman Coulter, UK). A sample specific 8bp index tag was incorporated during amplification to allow subsequent de-convolution of sample origin in all recurrent variants. Individual pools of normal and tumour samples were prepared and subjected to 454 pyrosequencing (Roche, Branford, CT, USA). Sequencing data were aligned as previously described and targeted evaluation of sequence reads by chromosome, position and variant base was performed to confirm somatic status of reported variant.
Papaemmanuil E., Rapado I., Li Y., Potter N.E., Wedge D.C., Tubio J., Alexandrov L.B., Van Loo P., Cooke S.L., Marshall J., Martincorena I., Hinton J., Gundem G., van Delft F.W., Nik-Zainal S., Jones D.R., Ramakrishna M., Titley I., Stebbings L., Leroy C., Menzies A., Gamble J., Robinson B., Mudie L., Raine K., O’Meara S., Teague J.W., Butler A.P., Cazzaniga G., Biondi A., Zuna J., Kempski H., Muschen M., Ford A.M., Stratton M.R., Greaves M, & Campbell P.J. (2014). RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nature genetics, 46(2), 116-125.
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4

Profiling Gut Microbiome Diversity in Poultry

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DNA extraction of pooled caecal digesta samples (maximum of 5 birds/pool) was performed using the DNAzol™ kit (Life Technologies Ltd., Paisley, UK). DNA pellets were re-suspended in 200 μl of TE elution buffer (Qiagen, UK) with concentration and purity assessed by Nanodrop. Bacterial DNA was analysed by 454-pyro sequencing (Roche, Indianapolis, U.S.A) targeting the V4-V5 region of the bacterial 16S ribosomal gene as a service by the Animal Health and Veterinary Laboratories Agency (AHVL), Weybridge, U.K. Sequencing returned over 10,000 reads per sample, each read was 400–500 bp in length and any poor quality reads were discarded. Methods used for prediction of identities from the sequencing data were automated and involved using BLASTn against Ribosomal databases [22 (link)]. Data were formatted by AHVL to show percentage abundance of each identified genus in relation to the overall microbiome.
Cadwell K., Niranji S.S., Armstrong V.L., Mowbray C.A., Bailey R., Watson K.A, & Hall J. (2017). AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut. BMC Genomics, 18, 637.
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5

16S rRNA Gene Profiling of Rhizosphere Soil

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Purified DNA extracts from rhizosphere soil samples were initially submitted to Macrogen, Inc. (Seoul, South Korea) for 454-pyrosequencing (Roche) using 16S rRNA as a gene target. The selection of primer set and sequencing of 16S rRNA gene libraries were according to the Macrogen, Inc. protocol and recommended for Roche 454 GS-FLX System using Titanium Chemistry (454 Life Sciences). Briefly, 16S rRNA gene libraries were prepared by PCR using the universal bacterial primer UNI_AMP-27F (5′-Zxxx GAG TTT GAT CMT GGC TCA G-3′ and UNI_AMP-518R (5′-K WTT ACC GCG GCT GCT GG-3′) (Lee et al., 2010 (link)), where Z and K represent two pyrosequencing primers (CCATCT CAT CCC TGC GTG TCT CCG ACT CAG and CCTATC CCC TGTG TGC CTT GGC AGT CTC AG), and xxx was designed for the sample identification barcoding key. The PCR reaction was as follow: a hot start at 95°C for 3 min, PCR amplification was carried out for 35 cycles at 94°C for 15 s, 55°C for 45 s, and 72°C for 1 min. A final extension step was carried out at 72°C for 8 min. The 16S rRNA gene libraries were sequenced by with Roche 454 GS-FLX System using Titanium Chemistry (Roche Diagnostics Corporation, Life Sciences, Branford, CT, United States).
Astorga-Eló M., Zhang Q., Larama G., Stoll A., Sadowsky M.J, & Jorquera M.A. (2020). Composition, Predicted Functions and Co-occurrence Networks of Rhizobacterial Communities Impacting Flowering Desert Events in the Atacama Desert, Chile. Frontiers in Microbiology, 11, 571.
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6

Profiling Lymphocyte Repertoire by LAM-PCR and NGS

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LAM-PCR and/or nrLAM-PCR were performed as described previously [34 (link), 35 (link)]. PCR amplicons of pretransplant and primary mice were sequenced with 454 pyrosequencing (Roche) as previously described [34 (link), 36 (link)] whereas those of secondary recipients were sequenced on the MiSeq instrument (Illumina) after sample preparation for high-throughput sequencing. Therefore, an additional PCR with special fusion-primers carrying MiSeq specific sequencing adaptors was performed. DNA barcoding was used for sequencing of multiple samples in a single run.
Negre O., Bartholomae C., Beuzard Y., Cavazzana M., Christiansen L., Courne C., Deichmann A., Denaro M., de Dreuzy E., Finer M., Fronza R., Gillet-Legrand B., Joubert C., Kutner R., Leboulch P., Maouche L., Paulard A., Pierciey FJ J.r., Rothe M., Ryu B., Schmidt M., von Kalle C., Payen E, & Veres G. (2015). Preclinical Evaluation of Efficacy and Safety of an Improved Lentiviral Vector for the Treatment of β-Thalassemia and Sickle Cell Disease. Current Gene Therapy, 15(1), 64-81.
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7

454 Pyrosequencing of Normalized cDNAs

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For 454 pyrosequencing (Roche Applied Science), 3 μg of normalized cDNAs was sent to the Core for Applied Genomics and Ecology (CAGE) facility at the University of Nebraska-Lincoln. The sequences obtained were preprocessed by filtering reads with low qualities (Q15) that were less than 100 bp as well as trimming SMART adapters and Ns. Finally, processed reads were clustered using the MIRA 3.4.0 assembler.
Valencia A., Wang H., Soto A., Aristizabal M., Arboleda J.W., Eyun S.I., Noriega D.D, & Siegfried B. (2016). Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts. PLoS ONE, 11(3), e0151001.
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8

Fab Phage-Display Library Analysis

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All experiments were performed with a previously described Fab phage-display library11 (link) based on the pComb3X vector.3 The library was deeply sequenced 5 times, in the Illumina MiSeq platform, and in a single experiment with 454 pyrosequencing (Roche). For each sequencing experiment, VH and VL amplicons were obtained as described above. The NGS raw data are shown in Supplementary Table S1.
Maranhão A.Q., Silva H.M., da Silva W.M., França R.K., De Leo T.C., Dias-Baruffi M., Burtet R.T, & Brigido M.M. (2020). Discovering Selected Antibodies From Deep-Sequenced Phage-Display Antibody Library Using ATTILA. Bioinformatics and Biology Insights, 14, 1177932220915240.
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9

Comparative Genomic Analysis of Sporothrix spp.

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Sporothrix schenckii and S. brasiliensis genomes were sequenced using next generation 454 pyrosequencing (Roche). Shotgun and paired-end 3 kb inserts libraries were constructed and sequenced in the 454 GS FLX platform according to Roche’s protocols at the Computational Genomics Unity of the National Laboratory for Scientific Computing (LNCC, Petrópolis, RJ, Brazil). Genomic assemblies were carried out using Newbler and Celera Assembler. Sequence gap filling and the removal of contigs corresponding to rDNA genes were manually done, decreasing the numbers of scaffolds and contigs. The assembled scaffolds generated by the two species were aligned and oriented using MAUVE [107 (link)]. Similarity scores and dot-plot graphs were generated using LALING/PLALING (http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=lalign).
Teixeira M.M., de Almeida L.G., Kubitschek-Barreira P., Alves F.L., Kioshima É.S., Abadio A.K., Fernandes L., Derengowski L.S., Ferreira K.S., Souza R.C., Ruiz J.C., de Andrade N.C., Paes H.C., Nicola A.M., Albuquerque P., Gerber A.L., Martins V.P., Peconick L.D., Neto A.V., Chaucanez C.B., Silva P.A., Cunha O.L., de Oliveira F.F., dos Santos T.C., Barros A.L., Soares M.A., de Oliveira L.M., Marini M.M., Villalobos-Duno H., Cunha M.M., de Hoog S., da Silveira J.F., Henrissat B., Niño-Vega G.A., Cisalpino P.S., Mora-Montes H.M., Almeida S.R., Stajich J.E., Lopes-Bezerra L.M., Vasconcelos A.T, & Felipe M.S. (2014). Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis. BMC Genomics, 15, 943.
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10

16S rRNA Gene Sequencing Using Pyrosequencing

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Bacterial 16S rRNA gene sequences were amplified and sequenced with Roche 454 pyrosequencing. All sequencing runs included appropriate extraction controls and sequencing (not template added) controls (Additional file 8). Amplicons were produced utilizing 338F and 906R primers adapted for the GS FLX Titanium pyrosequencing platform (Roche) to target the V2-V4 region of the 16S rRNA gene. The primers incorporated either the A linker, key, and a 10-nucleotide barcode (forward primer) or the B linker and key (reverse primer), followed by a sequence targeting a conserved region of the bacterial 16S rRNA gene. All individual PCR amplicons were purified, quantified, and pooled in equimolar ratios, and the library pool was gel purified prior to submission for sequencing as described in [45 (link)] with the exception that 30 PCR cycles were done. Sequencing was performed on the Roche/454 GS FLX+ System.
Rogers M.B., Firek B., Shi M., Yeh A., Brower-Sinning R., Aveson V., Kohl B.L., Fabio A., Carcillo J.A, & Morowitz M.J. (2016). Disruption of the microbiota across multiple body sites in critically ill children. Microbiome, 4, 66.
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