The normalized cDNA library was sequenced using Roche 454 GS-FLX pyrosequencing [29 ] with several modifications that improved sequence reproducibility while reducing labor-intensive sample manipulations [30 , 31 ]. Flowgrams from the GS-FLX were assembled by Newbler Assembler (Roche Applied Science). Contiguous sequences (contigs) and singletons were searched against the NCBI Genbank protein database using blastx where E ≤ 10-5 was considered a significant match to an expressed sequence tag (EST). Gene targets were annotated where possible with identity and functionality using Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/ ) and EuKaryotic Orthologous Groups (KOG, http://genome.jgi.doe.gov/Tutorial/tutorial/kog.html ) databases.
454 gs flx pyrosequencing
Manufactured by Roche
Sourced in Japan
The 454 GS-FLX pyrosequencing system is a next-generation DNA sequencing platform developed by Roche. It utilizes a non-optical, bioluminescence-based detection method to determine the sequence of DNA samples. The system is capable of generating high-throughput sequencing data with a focus on long read lengths.
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Lab products found in correlation
2 protocols using 454 gs flx pyrosequencing
1
Roche 454 Pyrosequencing of Normalized cDNA Library
2
Profiling Fungal Diversity via Pyrosequencing
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Universal primers NL1 and NL4 containing A and B sequencing adaptors were used to PCR-amplify DNA encoding the D1/D2 regions of the large subunit(LSU)of rRNA 4) . The primers used were:
B-NL1(5′ -cctatcccctgtgtgccttggcagtctcaGCATA TCAATAAGCGGAGGAAAAG-3′ ; the B adaptor is in lowercase)and A-NL4(5′ -ccatctcatccctgcgtg tctccgactcagatcagacacgNNNNNGGTCCGTGTTT CAAGACGG-3′ ; the A adaptor is in lowercase and the Ns are barcodes unique to each sample) . Following purification of the PCR amplicons, equimolar amounts were mixed in a single tube and subjected to 454 GS FLX pyrosequencing (Roche Diagnostics Japan, Tokyo, Japan) according to the manufacturerʼs instructions.
Sequence processing and data analysis Primer and barcode sequences were removed from the data and possible chimeras were excluded from analysis. Sequences ≥ 400 bp in length were analyzed. D1/D2 LSU sequences were identified using the RDP classifier(http: //rdp.cme.msu.edu) . The R package vegan［http: //cran.r-project.org/package = vegan］was used to construct a Shannon diversity index boxplot. Significance was tested using the one-tailed ttest with two-sample variance. A threedimensional principal coordinate analysis(PCoA) plot was normalized using the weighted values (http://www.qiime.org) .
B-NL1(5′ -cctatcccctgtgtgccttggcagtctcaGCATA TCAATAAGCGGAGGAAAAG-3′ ; the B adaptor is in lowercase)and A-NL4(5′ -ccatctcatccctgcgtg tctccgactcagatcagacacgNNNNNGGTCCGTGTTT CAAGACGG-3′ ; the A adaptor is in lowercase and the Ns are barcodes unique to each sample) . Following purification of the PCR amplicons, equimolar amounts were mixed in a single tube and subjected to 454 GS FLX pyrosequencing (Roche Diagnostics Japan, Tokyo, Japan) according to the manufacturerʼs instructions.
Sequence processing and data analysis Primer and barcode sequences were removed from the data and possible chimeras were excluded from analysis. Sequences ≥ 400 bp in length were analyzed. D1/D2 LSU sequences were identified using the RDP classifier(http: //rdp.cme.msu.edu) . The R package vegan［http: //cran.r-project.org/package = vegan］was used to construct a Shannon diversity index boxplot. Significance was tested using the one-tailed ttest with two-sample variance. A threedimensional principal coordinate analysis(PCoA) plot was normalized using the weighted values (http://www.qiime.org) .
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