R. albus SY3 was sequenced at the W.M. Keck Center for Comparative and Functional Genomics (University of Illinois at Urbana-Champaign). Total sequence data was generated from both a paired-ended 500-nt insert library sequenced on a single lane of HiSeq (Illumina) and a paired ended 3-kb insert library sequenced on a full plate of 454 sequencing (Roche Diagnostics). These approaches yielded 47 million 100-nt reads (4.7 billion bases) and 1.4 million reads with an average read length of 402 nt (577 million bases; 71% true paired end, actual paired distance was 2386+597 nt), respectively. The 454 sequence data was assembled using Newbler v2.5.3 and the Illumina was assembled using Velvet v1.1. The assemblies were combined using Minimus2. The sequence assembled to 4 scaffolds (N50 = 1,120,630 bp) and 97 contigs (N50 = 114,193). 99.95% of bases were >Q40 and all others (1808 bp) were Q39. The total sequence produced was 3,832,777 nt and the genome was estimated to be 4.1 Mb, giving us 93.5% coverage. The modal sequence coverage depth was 131×. The sequence was annotated using subsystems in RAST.
454 sequencing
Manufactured by Roche
Sourced in United States
The 454 sequencing is a high-throughput DNA sequencing technology developed by Roche. It utilizes a technique known as pyrosequencing to determine the nucleotide sequence of a DNA sample. The 454 sequencing system is designed to generate large amounts of sequence data in a relatively short timeframe.
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Lab products found in correlation
Picogreen, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Proteome discoverer v2, by Thermo Fisher Scientific (1 mentions)
Mascot search, by Matrix Science (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 1.0 instrument, by Roche (1 mentions)
Lightcycler faststart dna master sybr green 1 kit, by Roche (1 mentions)
Dneasy 96 kit, by Qiagen (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Dcode system, by Bio-Rad (1 mentions)
Biotaq polymerase, by Meridian Bioscience (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Ez dna methylation direct kit, by Zymo Research (1 mentions)
Solexa, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
15 protocols using 454 sequencing
1
Sequencing and Assembly of Ruminococcus albus SY3
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R. albus SY3 was sequenced at the W.M. Keck Center for Comparative and Functional Genomics (University of Illinois at Urbana-Champaign). Total sequence data was generated from both a paired-ended 500-nt insert library sequenced on a single lane of HiSeq (Illumina) and a paired ended 3-kb insert library sequenced on a full plate of 454 sequencing (Roche Diagnostics). These approaches yielded 47 million 100-nt reads (4.7 billion bases) and 1.4 million reads with an average read length of 402 nt (577 million bases; 71% true paired end, actual paired distance was 2386+597 nt), respectively. The 454 sequence data was assembled using Newbler v2.5.3 and the Illumina was assembled using Velvet v1.1. The assemblies were combined using Minimus2. The sequence assembled to 4 scaffolds (N50 = 1,120,630 bp) and 97 contigs (N50 = 114,193). 99.95% of bases were >Q40 and all others (1808 bp) were Q39. The total sequence produced was 3,832,777 nt and the genome was estimated to be 4.1 Mb, giving us 93.5% coverage. The modal sequence coverage depth was 131×. The sequence was annotated using subsystems in RAST.
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R. albus SY3 was sequenced at the W.M. Keck Center for Comparative and Functional Genomics (University of Illinois at Urbana-Champaign). Total sequence data was generated from both a paired-ended 500-nt insert library sequenced on a single lane of HiSeq (Illumina) and a paired ended 3-kb insert library sequenced on a full plate of 454 sequencing (Roche Diagnostics). These approaches yielded 47 million 100-nt reads (4.7 billion bases) and 1.4 million reads with an average read length of 402 nt (577 million bases; 71% true paired end, actual paired distance was 2386+597 nt), respectively. The 454 sequence data was assembled using Newbler v2.5.3 and the Illumina was assembled using Velvet v1.1. The assemblies were combined using Minimus2. The sequence assembled to 4 scaffolds (N50 = 1,120,630 bp) and 97 contigs (N50 = 114,193). 99.95% of bases were >Q40 and all others (1808 bp) were Q39. The total sequence produced was 3,832,777 nt and the genome was estimated to be 4.1 Mb, giving us 93.5% coverage. The modal sequence coverage depth was 131×. The sequence was annotated using subsystems in RAST.
2
Profiling Immunoglobulin Repertoire via NGS
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In brief, Total RNA was isolated from PBMC (RNeasy mini kit, Qiagen) and RNA quality was assessed using an Agilent Bioanalyzer. Total isolated RNA was reverse transcribed (SMARTerTM RACE, Clontech), and approximately 27% of each cDNA reaction was used for IgG-VH amplification via PCR using SMARTerTM RACE 10X Universal primer mix (Clontech) and an IgG specific 3′ primer (5′-GGG AAG ACS GAT GGG CCC TTG GTG G-3′) for 31 cycles following the manufacturer’s recommendation. In addition to the IgG-specific portion, reverse primers also contained the Lib-L specific adaptor (454 sequencing, Roche) and barcode sequences. Barcoded IgM-VH (~715 bp) and IgG-VH (~640 bp) transcript libraries were purified using AMPure XP (Beckman Coulter Inc.), quantified using PicoGreen (Life Technologies) and normalized to 1 × 109 molecules/μl. Uniquely barcoded samples were combined in pools of normalized IgG-VH amplified libraries and subjected to emulsion PCR and unidirectional sequencing using the GS FLX Titanium Lib-L chemistry (454 sequencing, Roche).
3
Genome Sequencing and Annotation of Fungus D263
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About 400 μg isolated genomic DNA was used for library preparation, and 454 sequencing (Roche, Switzerland) was used to acquire sequence reads with average length of 373 bp. The draft genome of D263 was assembled by using Newbler V2.3, with mapping statistics shown in Additional file 1 : Table S1. Blast2Go was used to identify enriched Gene Ontology (GO) terms in D263 genes [58 (link)]. GOBU was used to characterize the GO terms in the D263 genome [59 ]. KofamScan was used to predict the KEGG pathways of D263 genes [60 (link)]. The mapping statistics are shown in Additional file 6 : Table S5. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank (Accession no. JAAOZU000000000). The GO terms and CAZy enzymes in T. reesei V2.0 and A. niger ATCC1015 were searched in the Joint Genome Institute (JGI) MycoCosm database [31 (link), 61 (link)–63 (link)]. Hierarchical analysis was conducted based on Pearson correlation. For proteome analyses, protein bands showing cellulolytic enzyme activities were extracted from SDS-PAGE gels. The gel slices were trypsin-digested and underwent mass spectrometry. The amino acid sequences of annotated D263 genes underwent a Mascot search (Matrix Science, USA). Protein abundance was calculated by using ProteomeDiscoverer v2.4 (Thermo-Fisher, USA).
4
Quantifying Amplicon Concentration for 454 Sequencing
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In preparation for 454 sequencing (Roche), the concentration of purified amplicons obtained by micPCR and traditional PCR was measured using a 16 S quantitative PCR (qPCR). The qPCR reactions were performed in 10 μL reaction volumes using the LightCycler FastStart DNA Master SYBR Green I Kit (Roche) with the addition of 0.5 μM of amplification primer 357F and 926R without the universal tails. The PCR reactions were performed on a LightCycler 1.0 instrument (Roche), under the following conditions: initial denaturation at 95 °C for 10 minutes followed by 45 cycles of PCR, with cycling conditions of 1 second at 95 °C, 5 seconds at 55 °C, and 30 seconds at 72 °C. The concentration of purified amplicons obtained by micPCR and traditional PCR were normalized to 1E + 05 molecules/μL using a serial dilution of a standard solution containing 16S rRNA genes derived from a highly bacterial diverse sludge sample that was calibrated using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies).
5
Mutational Profiling of TP53 in Tumor Cell Lines
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Genomic DNA was extracted from frozen cell pellets of each tumor cell line according to the DNeasy 96 kit protocol (QIAGEN) and sequenced for the presence of mutations in TP53, either using 454 sequencing (Roche Diagnostics) or WAVE® Mutation Detection System and SURVEYOR® Nuclease technology (Transgenomic, Inc.; [27 (link)]).
Additional data regarding the p53 mutational status of tumor cell lines in the panel were obtained from the Wellcome Trust Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC v44–62 releases),http://www.sanger.ac.uk/cosmic [11 (link), 28 ]; the IARC TP53 database (version R15), p53.iarc.fr/TP53GeneVariations.aspx [29 (link)], and the TP53 Mutation Database, http://www.p53.fr [18 (link)].
Additional data regarding the p53 mutational status of tumor cell lines in the panel were obtained from the Wellcome Trust Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC v44–62 releases),
6
Transcriptome Sequencing of Equisetum fluviatile
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Lateral shoot tissue (approximately 500 mg) of a single E. fluviatile individual was finely ground in liquid N2 and mixed with 3 ml TRIzol (Invitrogen; www.lifetechnologies.com/ ). The suspension was centrifuged at 12 000 g (4°C, 5 min), and extracted with 0.2 volumes of chloroform. The aqueous phase was mixed with 0.54 volumes of ethanol and RNA purified from it by use of a PureLink RNA Mini Kit (Life Technologies; www.lifetechnologies.com ). cDNA was synthesized and normalized with Mint and Trimmer kits (Evrogen; www.evrogen.com/ ) and used for 454 sequencing (Roche; www.454.com ) at Edinburgh Genomics ( https://genomics.ed.ac.uk ). Raw data were assembled with Roche Newbler assembler version 2.5 (268 000 reads; length 249 ± 111 nucleotides; mean ± SD).
7
Profiling Gut Bacterial Communities in Mice
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Bacterial communities were profiled in the mouse caecum using Denaturing Gradient Gel Electrophoresis (DGGE), 454 sequencing (Roche, USA), and Illumina MiSeq (USA). DGGE assessment of the bacterial communities was as follows: PCR amplification of the 16S rRNA gene used universal primers 341F-GC and 518R (Muyzer et al., 1993 (link)), and reaction conditions: 5U BioTaq polymerase in 1X buffer (Bioline, UK), 1.5 mM MgCl2, 20 pmol primers, 0.2 mM dNTPs, 5 μg BSA, and 10–50 ng of template DNA in a final volume of 50 μL. The cycle sequence consisted of initial denaturation step of 95 °C 5 min, then 30 cycles of 95 °C 1 min; 55 °C 1 min; 72 °C 1 min, and final extension of 72 °C 10 min. PCR products were purified (QiaGEN Minelute kit) before loading onto a DGGE gel (150 ng/lane). Samples were separated using the D-code system (Bio-Rad, USA) on 10% w/v acrylamide gel with a gradient of 30–70% denaturant at 60 °C for 16 h at 63 V. Gels were stained for 30 min using SYBR Gold (Invitrogen, USA). DGGE Gels were analysed with Phoretix 1D Advanced gel analysis software (Ver. 5.0, Nonlinear Dynamics Ltd.), with binary matrix of band presence/absence of individual bands used for sample comparison.
8
Bisulfite Conversion and Methylation Analysis
9
Identification of HBV Splice Variants
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Four pooled PCR amplified products (NVRs-gtB, NVRs-gtC, EVRs-gtB, EVRs-gtC) containing ~200–800 bp DNA fragments were prepared as shown in Fig. S4 . The amplicons were purified with the microconcolumns (Qiagen) before sequencing, the products of which were used to generate multiplex identifier (MID)-tagged libraries for 454 sequencing (Roche) according to the instructions of the manufacturer. Sequence reads from the 454/Roche GS Junior sequencer were subjected for performing BLAST with the reference sequences of HBV (2811–611) for identifying the spliced accept and donor site(s) of each read, according to which the reads were then classified into a number of species of spHBV variants. Only the species of spHBV that had a proportion of at least 0.01% among the total reads were calculated for analyzing the distribution of the spHBV variants.
10
Amphioxus Transcriptome Profiling
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Chinese amphioxus, B. belcheri, were collected and reared in Beihai, Guangxi, China. The mRNAs from male adult amphioxus were extracted for Roche 454 sequencing (Lib-1). Two cDNA libraries were constructed from total RNAs of samples from different developmental stages, including fertilized egg, 2-cell, 16-cell, 64-cell, 128–256-cell, gastrula, neurula, hatch, 12 hours, 24 hours, 48 hours, male adult and female adult stages and were sequenced with Illumina Solexa paired-end sequencing (Lib-2 with normalization and Lib-3 without normalization). The RNA libraries from each stage were also sequenced through DGE-Seq by BGI as described in the Illumina DGE-Seq protocol. All sequencing data were submitted to the National Center for Biotechnology Information (NCBI) (BioProject: PRJNA310680).
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