Pyrosequencing of the cDNA library was performed by the Beijing Autolab Biotechnology Company using a 454 GS-FLX sequencer (Roche, IN, USA) according to the manufacturer’s instructions. All sequencing reads were deposited into the Short Read Archive (SRA) of the National Center for Biotechnology Information (NCBI), and can be accessed under the accession numbers SRR838973 and SRR838974 for the male and female antennal transcriptomes, respectively.
454 gs flx sequencer
Manufactured by Roche
Sourced in United States, Germany
The 454 GS-FLX sequencer is a next-generation DNA sequencing system developed by Roche. It utilizes pyrosequencing technology to enable rapid and high-throughput sequencing of DNA samples. The core function of the 454 GS-FLX sequencer is to perform DNA sequencing, providing researchers with genomic data for various applications.
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Lab products found in correlation
Hiseq 2000, by Illumina (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rneasy plant midi kit, by Qiagen (1 mentions)
Titanium reagents, by Roche (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Access array system, by Standard BioTools (1 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Pfu polymerase, by New England Biolabs (1 mentions)
Seqman, by DNASTAR (1 mentions)
Gs junior sequencer, by Roche (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Rnaprotect bacteria reagent, by Qiagen (1 mentions)
Titanium chemistry, by Roche (1 mentions)
Phusion hot start high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Ampure beads, by Agilent Technologies (1 mentions)
Rapid library preparation kit, by Roche (1 mentions)
Tbs 380 fluorometer, by Promega (1 mentions)
Dsdna br assay, by Thermo Fisher Scientific (1 mentions)
25 protocols using 454 gs flx sequencer
1
Antennal Transcriptome Sequencing
2
RNA Extraction and Normalization for Transcriptome Sequencing
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Total RNA was extracted from entire shoots, including leaves and traps, from all three sources described above. Inflorescences were not used in this study. Approximately 800 mg of fresh weight (2 individuals) of whole shoots in three replicates from each source were ground in liquid nitrogen using a mortar and pestle and RNA was extracted using RNeasy Plant Midi Kit (Qiagen Inc., Valencia, CA, USA). The results of the parallel extractions were pooled in equal proportions into a single sample and the quality and quantity of the extracted RNA were verified by gel electrophoresis and by spectrophotometer, measuring the 230/260 ratio with Biophotometer (Eppendorf, Germany).
A pooled RNA sample was provided to GATC Biotech (Konstanz, Germany) for the construction of a cDNA library and subsequent sequencing. Library construction involved DNAselection for polyadenylated (polyA+) transcripts to enrich for protein-coding mRNAs, DNase I treatment and normalization through denaturation/reassociation of cDNA, to improve the representation of low-copy transcripts and thereby maximize gene discovery. The resulting library was sequenced with a full picotiter plate on a 454 GS-FLX sequencer with Titanium reagents (Roche Applied Science, Indianapolis, IN, USA), using standard 454 protocols.
A pooled RNA sample was provided to GATC Biotech (Konstanz, Germany) for the construction of a cDNA library and subsequent sequencing. Library construction involved DNAselection for polyadenylated (polyA+) transcripts to enrich for protein-coding mRNAs, DNase I treatment and normalization through denaturation/reassociation of cDNA, to improve the representation of low-copy transcripts and thereby maximize gene discovery. The resulting library was sequenced with a full picotiter plate on a 454 GS-FLX sequencer with Titanium reagents (Roche Applied Science, Indianapolis, IN, USA), using standard 454 protocols.
3
Targeted Sequencing of Salmonella Virulence Genes
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For targeted massive parallel sequencing, primer pairs for 15 genes, including the genes for 12 fimbrial adhesins (StcD, SafD, BcfD, FimH, StbD, SthE, StdD, StiH, StfH, LpfD, StjA, PefA)21 (link) and 3 outer-membrane proteins (OmpA, OmpC, OmpN) were designed and synthesized (Integrated DNA Technologies, Inc.) with 3–4 primer pairs per gene (Supplementary Table 6 ). The sequencing libraries were prepared using the Access Array system (Fluidigm South San Francisco)14 (link). Quality and quantity of the amplicon libraries were evaluated with a 2,100 Bioanalyzer instrument (Agilent Technologies) and NanoDrop. The libraries were pooled in equal amounts for pyrosequencing with a 454 GS FLX sequencer using Titanium chemistry (454 Life Sciences, Roche) at the DNA Sequencing Facility of UPENN. An in-house Perl script was used for sequence splitting and barcode removal. Sequence assembly and mapping were done with SeqMan (DNASTAR, Inc.). A total of 15 genes of 382 strains were sequenced with a coverage of more than 30 and a Phred quality score of more than 40 for data analysis. For Sanger DNA sequencing, the fimH gene of 210 clinical isolates from various S. enterica serovars were amplified with the Pfu polymerase (New England Biolabs Inc.) and each individual gene sequence was assembled using at least three sequencing reads to get a Phred quality score of more than 30.
4
Genome Assembly of Lactobacillus reuteri
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For the initial draft assembly [26 (link)] L. reuteri DNA had been used to generate in excess of 365 Mbp of sequence from a combination of shotgun and 3-kbp paired-end libraries (220 Mbp and 145 Mbp, respectively) on the 454 GS FLX sequencer (Roche) using the Titanium Chemistry. Reads passing the default filter settings had been assembled using gsAssembly V2.3 software (Roche) and had generated 13 scaffolds containing 99 large contigs (>500 bp) and spanning 1.96 Mbp of sequence. Standard PCR followed by primer walk sequencing on the resulting products was used to close the gaps located in scaffolds. Multiplex PCR was employed to identify adjoining contigs and respective primer pairs for which no linkage had been established previously and upon re-amplification under standard conditions the resulting products were analysed by primer walk sequencing. The sequence assembly was carried out using the Phred/Phrap [75 (link)] software in conjunction with the Staden package [76 (link)] and the expected error rate is around 1/500000 bp.
5
454 Sequencing of GcM5-1A Genome
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The GcM5-1A genome was sequenced on a 454 GS-FLX sequencer (Roche, Inc, Basel, Switzerland.) using the FLX Titanium Sequencing Kit (Roche, Inc, Basel, Switzerland.). Sequence assembly was conducted with a Newbler de novo Assembler (version 2.5.3) with default parameters. In the final assembly, contigs less than 100 bp in length were discarded. Sequence data from this study were deposited at DDBJ/EMBL/GenBank under accession number JJOE00000000 .
6
16S rRNA Microbiome Profiling of Armadillidium
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The 16S rRNA gene amplicon sequences analysed in this study derive from the same pyrosequencing dataset used for the initial characterization of the microbiota of A. vulgare in different host tissues and populations75 (Accession Number PRJEB8160). Briefly, a 526 bp-fragment spanning the variable regions V1-V3 of the 16S rRNA gene was amplified using the universal primers 27 F and 520 R. Primers were adapted for 454 pyrosequencing by adding the 454 Adapter A and a 10-bp Multiplex Identifier sequence (MID) to the reverse primer 520 R as well as the 454 Adapter B to the forward primer 27 F. Amplicons were obtained from five different tissues (haemolymph, nerve cord, gonads, midgut caeca and hindgut) and up to 10 biological replicates from the same tissue and sample type (origin x gender x Wolbachia strain) were pooled for sequencing, resulting in 55 amplicon pools (see Supplementary Table S1 for details). The amplicon pools were purified using AMPure Beads (Agencourt Bioscience Corporation), quantified using PicoGreen (Invitrogen) and sequenced on a 454 GS FLX sequencer (Roche, 454 Life Sciences) by GenoScreen (Lille, France) as well as on a GS Junior sequencer (Roche, 454 Life Sciences) at the University of Poitiers (France).
7
Genome Sequencing of Bradyrhizobium sp. SUTN9-2
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Cells of Bradyrhizobium sp. SUTN9-2 were cultured at 30°C in HM salt medium (5 (link)). In the stationary phase, cells were harvested by centrifugation, and total DNA was prepared as previously described (17 ). The 8-kb paired-end 454 library of SUTN9-2 was constructed from total DNA by Takara Bio (Shiga, Japan). Genome sequencing was performed using a 454 GS FLX+ sequencer (Roche Diagnostics K.K.; Tokyo, Japan). Generated sequences were assembled using Roche GS De Novo Assembler version 2.8 (Roche Diagnostics K.K.; Tokyo, Japan). Predictions of gene regions and annotation were performed using MiGAP (http://www.migap.org/index.php/en/about-migap ). A circular genome map showing the GC skew and GC content was created using the GCview Server (http://stothard.afns.ualberta.ca/cgview_server ).
The genome sequences of Bradyrhizobium sp. SUTN9-2 (accession number LAXE00000000) were compared with the whole genome of B. diazoefficiens USDA110 (33 (link)) using the program GenomeMatcher (21 (link)) at the amino acid level. The annotated genome sequences of USDA110 (accession number BA000040) were obtained from Genome Assembly/Annotation Projects (NCBI database). The sequences of chromosome and plasmid pDOA9 are available in the DDBJ/GenBank/EMBL database (accession numbers DF820425 and DF820426, respectively).
The genome sequences of Bradyrhizobium sp. SUTN9-2 (accession number LAXE00000000) were compared with the whole genome of B. diazoefficiens USDA110 (33 (link)) using the program GenomeMatcher (21 (link)) at the amino acid level. The annotated genome sequences of USDA110 (accession number BA000040) were obtained from Genome Assembly/Annotation Projects (NCBI database). The sequences of chromosome and plasmid pDOA9 are available in the DDBJ/GenBank/EMBL database (accession numbers DF820425 and DF820426, respectively).
8
Bacterial Diversity Analysis via 16S rRNA Sequencing
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For bacterial diversity analysis, hypervariable regions 3–5 (V3–V5) of the 16S rRNA gene were used to generate PCR amplicons, as described by Herlemann et al. (2011) (link), but with a modification of 30 PCR cycles. Sequencing was performed by Eurofins MWG GmbH using 454 GS-FLX sequencer (Roche). The denoising tool Acacia (Bragg et al., 2012 (link)) was used to correct amplicon pyrosequencing errors. Primer sequences were trimmed from the reads and the sequences were clipped 400 bp downstream of the primer. Reads shorter than 400 bp (excluding the primer) and/or containing Ns were excluded. Reads that were found only once in the sample set were removed from the analysis. Sequences were aligned and clustered at 97% identity into operational taxonomic units (OTUs), as described by Herlemann et al. (2011) (link), using the pyrosequencing pipeline at RDP (Cole et al., 2009 (link)). The online tool Decipher (Wright et al., 2012 (link)) was used to identify and remove chimeric sequences in the remaining OTUs. The abundances of the resulting OTUs were normalized using the relative proportions of individual OTU reads from all sample reads. Sequences have been deposited in the European Nucleotide Archive (ENA), with the study accession: http://www.ebi.ac.uk/ena/data/view/PRJEB8816 .
9
Tiger Shark DNA Sequencing and Microsatellite Isolation
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The total genomic DNA was extracted from each sample following the protocol described by Ivanova, Dewaard & Hebert (2006) (link). A sample (voucher: TIG03SP) of 100 μg of tiger-shark DNA from São Paulo coast (−25.1164, −47.6082) was sequenced on a Roche 454 GS FLX sequencer with Titanium platform “Genome sequencer 20 System” (Instituto Agrobiotecnológico de Rosário–INDEAR, Argentina), following procedures described in Margulies et al. (2005) (link).
To isolate microsatellites and design primers for population genetics all sequences of the SSR were compiled using Primer3 (Rozen & Skaletsky, 1999 ) and BatchPrimer3 (You et al., 2008 (link)). Primers were designed based on the following criteria: primer size of 20 bp (min = 18, max = 22 bp), ideal annealing temperature of 60 °C (min = 55 °C, max = 63 °C), GC optimum of 60% (min = 40%, max = 80%) and the size of the amplified product ranging from 50–500 bp. The sequences were then grouped and aligned in the Clustal W software (Thompson, Higgins & Gibson, 1994 (link)), identifying duplicated sequences for the same locus.
To isolate microsatellites and design primers for population genetics all sequences of the SSR were compiled using Primer3 (Rozen & Skaletsky, 1999 ) and BatchPrimer3 (You et al., 2008 (link)). Primers were designed based on the following criteria: primer size of 20 bp (min = 18, max = 22 bp), ideal annealing temperature of 60 °C (min = 55 °C, max = 63 °C), GC optimum of 60% (min = 40%, max = 80%) and the size of the amplified product ranging from 50–500 bp. The sequences were then grouped and aligned in the Clustal W software (Thompson, Higgins & Gibson, 1994 (link)), identifying duplicated sequences for the same locus.
10
Characterization of TpUB05 Gene Sequence
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TpUB05 recombinant gene construct was sequenced by pyrosequencing using the 454 GS FLX Sequencer (Roche) and analyzed with VecSreen (National Centre for Biotechnology Information; NCBI) to remove vector sequences. The TpUB05 sequence was then analysed with BLAST of the GenBank non-redundant nucleotide and protein databases (NCBI) using the family of algorithms with default parameters [20 (link)]. The open reading frame was determined by open reading frame (ORF)-Finder and conserved-domain search was performed using NCBI conserved domain search [21 (link)]. The significant match with TP04_0076 was then selected for further analyses. Predictions of the physicochemical and structural properties of the derived protein sequence were performed using the protein analysis software available at Expasy.
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