Gs junior titanium sequencing kit

Manufactured by Roche

Sourced in United States

The GS Junior Titanium Sequencing Kit is a laboratory equipment product designed for DNA sequencing. It provides the necessary components for performing DNA sequencing analysis using the GS Junior Sequencing System.

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9 protocols using gs junior titanium sequencing kit

Genomic DNA Sequencing and Microsatellite Enrichment

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The GS FLX Titanium Rapid Library Preparation Kit and the GS Junior Titanium Sequencing Kit (Roche 454 Life Sciences, Branford, Connecticut, USA) were used to generate a shotgun library of genomic DNA and sequencing on the Roche GS Junior 454 System (Roche 454 Life Sciences). The 454 pyrosequencing yielded a total of 56,545 sequence reads ranging from 40 to 1195 bp with an average length of 476 bp.
To obtain a larger number of polymorphic markers, we additionally developed and sequenced a microsatellite-enriched library. Size-selected genomic DNA fragments were enriched for microsatellite content by using magnetic streptavidin beads and biotin-labeled GATA, GTAT, AAAC, and AAAG repeat oligonucleotides. The enriched library was prepared and analyzed on a Roche 454 platform using the GS FLX Titanium reagents according to the manufacturer’s manual. This produced 14,467 sequence reads ranging from 80 to 550 bp with an average length of 459 bp.

Duwe V.K., Ismail S.A., Buser A., Sossai E., Borsch T, & Muller L.A. (2015). Fourteen polymorphic microsatellite markers for the threatened Arnica montana (Asteraceae). Applications in Plant Sciences, 3(1), apps.1400091.

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Targeted Sequencing of CRC-Associated Genes

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Using normal, benign and malignant colorectal tissue samples, mutation hot-spot regions of 12 CRC-associated genes (APC, B-Raf proto-oncogene, serine/threonine kinase (BRAF), catenin beta 1 (CTNNB1), epidermal growth factor receptor (EGFR), F-box and WD repeat domain containing 7 (FBXW7), KRAS proto-oncogene, GTPase (KRAS), NRAS proto-oncogene, GTPase (NRAS), mutS homolog 6 (MSH6), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), SMAD family member 2 and 4 (SMAD2 and SMAD4), tumor protein 53 (TP53)) were amplified using a custom-made multiplex PCR panel previously designed by our research group [27 (link)]. Amplicon sequencing was carried out on a GS Junior instrument (Roche) using ligated and barcoded adaptors as described earlier [27 (link)]. Bead enrichment and sequencing were performed using GS Junior Titanium Sequencing Kit (Roche) according to the Sequencing Method Manual, GS FLX Titanium Series. For variant identification, Amplicon Variant Analyzer software (Roche) was applied.

Molnár B., Galamb O., Péterfia B., Wichmann B., Csabai I., Bodor A., Kalmár A., Szigeti K.A., Barták B.K., Nagy Z.B., Valcz G., Patai Á.V., Igaz P, & Tulassay Z. (2018). Gene promoter and exon DNA methylation changes in colon cancer development – mRNA expression and tumor mutation alterations. BMC Cancer, 18, 695.

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HIV-1 V3 Region Amplification and Sequencing

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Prior to deep-sequencing, the V3 region was re-amplified in triplicate from all plasma and PBMC-derived nucleic acid extracts obtained from each individual. Nested second round amplification was performed using forward and reverse primers incorporating one of 12 multiplex identifier (MID) tags and a linker sequence at the 5′ end and visualized on a 1% agarose gel. Amplicons were quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) on a DTX 880 Multimode Detector (Beckman Coulter), pooled in equal proportions, purified, re-quantified, and deep-sequenced using the GS Junior Titanium Sequencing Kit on a GS Junior instrument (Roche/454). To avoid low-level, intra-run sequence cross-contamination by genetically similar amplicons, we sequenced each sample on a separate GS-Junior run (as each run typically included 24 V3 amplicons, this means that each run contained one amplicon from the present study and 23 V3 amplicons from patients unrelated to the present study). The one exception was sample +35M^Plasma/PBMC from the CCR5wt/wt individual, where data are derived from an initial run that included other study samples. Inclusion of data from this sample was deemed appropriate after quality-control experiments confirmed that HIV-1 sequences and their distributions obtained from separate vs. combined runs were highly concordant (not shown).

Le A.Q., Taylor J., Dong W., McCloskey R., Woods C., Danroth R., Hayashi K., Milloy M.J., Poon A.F, & Brumme Z.L. (2015). Differential evolution of a CXCR4-using HIV-1 strain in CCR5wt/wt and CCR5∆32/∆32 hosts revealed by longitudinal deep sequencing and phylogenetic reconstruction. Scientific Reports, 5, 17607.

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NGS Protocol for PKHD1 Gene Analysis

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The PKHD1 library for next-generation sequencing counted 87 amplicons, and was prepared in two steps with a universal-tailed amplicon sequencing design. Target-specific universal-tailed fusion primers (Generi Biotech) used for the first round of PCR have been described previously in the literature [15 (link)]. The library was purified by Agentcourt® AMPure® XP (Beckman Coulter), and the final library of 6 patients was mixed regarding their concentration (Quant-iT™ PicoGreen® dsDNA Assay Kit, Invitrogen), and then prepared for a sequencing run on a GS Junior with a GS Junior Titanium emPCR Kit (Lib-A) and a GS Junior Titanium Sequencing Kit (Roche Diagnostics). The data were analyzed using the Sequence Pilot program (JSI Medical Systems GmbH). All probable causal mutations found by NGS were confirmed in patients by Sanger sequencing done on an ABI PRISM 3130 Genetic Analyzer (Life Technologies); targeted Sanger sequencing was also performed in the parents/relatives of patients to determine the position of mutations at homologous chromosomes. Subsequent MLPA analysis (Multiplex ligation-dependent probe amplification) was done for all patients without 2 causal mutations found by NGS. The kits used for MLPA analysis were SALSA MLPA P341 PKHD1 mix 1, and P342-PKHD1 mix 2 (MRC Holland). The software used for the analysis of MLPA data was Coffalyser.Net (MRC Holland).

Obeidova L., Seeman T., Elisakova V., Reiterova J., Puchmajerova A, & Stekrova J. (2015). Molecular genetic analysis of PKHD1 by next-generation sequencing in Czech families with autosomal recessive polycystic kidney disease. BMC Medical Genetics, 16, 116.

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Genotypic HIV-1 Drug Resistance Monitoring

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The study was undertaken at the Virgen de la Victoria Hospital, a reference centre of the study of HIV-1 genotype drug resistance for six hospitals from the region of Malaga (southern Spain). A genotype resistance test has been routinely undertaken in our centre since 2004 for all the patients with confirmed HIV-1 infection at the time of diagnosis and before starting combination antiretroviral therapy (ART). A partial region of HIV-1 pol gene, encoding the complete protease (PR) and partial reverse transcriptase (RT), was sequenced using RT–PCR and Sanger sequencing (Trugene HIV Genotyping Kit®, Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) or 454 pyrosequencing (GS Junior Titanium Sequencing Kit® Roche Diagnostics Gmbh, Mannheim, Germany), depending on the date of sample collection (before or after 2014, respectively). The subtype for each FASTA sequence provided was assigned through REGA v3.0 and sequences determined as CRF19_cpx subtype, afterwards confirmed by phylogenetic analysis.

González-Domenech C.M., Viciana I., Delaye L., Mayorga M.L., Palacios R., de la Torre J., Jarilla F., Castaño M., del Arco A., Clavijo E, & Santos J. (2018). Emergence as an outbreak of the HIV-1 CRF19_cpx variant in treatment-naïve patients in southern Spain. PLoS ONE, 13(1), e0190544.

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Viral RNA Extraction and Rabies Genome Sequencing

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Viral RNA was extracted with the NucleoSpin® RNA kit (Macherey-Nagel) as per the manufacturer’s instructions and stored at –70°C until use. The first half of the rabies genome (positions 0–6433) was reverse transcribed from 1 μg of RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) with a mixture of all 4 reverse primers listed in Table 1. One μL of cDNA was used as a template for PCR using Q5^® High-Fidelity DNA Polymerase (New England BioLabs). The PCRs were designed so that four partially-overlapping fragments with 4 pairs of primers were produced. For the list of primers, see Table 1. The four PCR products obtained for each vaccine batch were pooled, fragmented and ligated with adaptors with different sequence modifications (bar codes) using the GS Rapid Library Prep Kit and GS Rapid Library MID Adaptor Kit (Roche). This enabled the post-sequencing identification of each vaccine batch sequence after a single sequencing run. Following emulsion PCR using the GS Junior Titanium Lib-L emPCR Kit, the pyrosequencing was performed using the GS Junior Titanium Sequencing Kit according to the instructions of the manufacturer and the 454 GS Junior sequencer (Roche). Data were analyzed by the Reference Mapper included in the 454 GS Junior software using the SAD Bern genomic sequence as a reference (GenBank accession number EF206708).

Cliquet F., Picard-Meyer E., Mojzis M., Dirbakova Z., Muizniece Z., Jaceviciene I., Mutinelli F., Matulova M., Frolichova J., Rychlik I, & Celer V. (2015). In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife. PLoS ONE, 10(10), e0141537.

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Microbial Diversity Analysis by Sequencing

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Purified PCR products were used to construct gene libraries and determine nucleotide sequences using a GS Junior Titanium Sequencing kit (Roche Diagnostics, Indianapolis, IN, USA). Sequence reads obtained by pyrosequencing were analyzed as previously described (Kim et al. 2012 (link); Diouf et al. 2015 ). Sequences obtained with GS Junior were compared with the 18S rDNA gene sequences from the non-redundant sequence database using the NCBI Basic Local Alignment Search Tool (BLAST; v.2.2.30+) for taxonomic identification and analysis of microbial diversity. Each sequence was then classified taxonomically using NCBI Metagenome Analyzer (MEGAN; v.5.10.1), and the results were used for taxonomic classification. Isolates were classified according to the hierarchy of super-kingdom, kingdom, phylum, class, order, family, genus, and species. A rooted phylogenetic tree was generated by assigning all sequences to each node. Each group of identified microorganisms is presented according to the Integrated Taxonomic Information System (ITIS) taxonomic counting heat maps. Microsoft Excel (Redmond, WA, USA) was used for data analysis.

Kang Y.M., Choi J.E., Komakech R., Park J.H., Kim D.W., Cho K.M., Kang S.M., Choi S.H., Song K.C., Ryu C.M., Lee K.C, & Lee J.S. (2017). Characterization of a novel yeast species Metschnikowia persimmonesis KCTC 12991BP (KIOM G15050 type strain) isolated from a medicinal plant, Korean persimmon calyx (Diospyros kaki Thumb). AMB Express, 7, 199.

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FV3 Genome Sequencing Protocol

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Standard kits and protocols developed by the manufacturer were used to sequence the SSME sample on a 454 GS-FLX platform (Roche Diagnostics Corporation). Briefly, a Rapid Library Preparation Kit (Roche, Mississauga, ON) was used to mechanically shear 500 ng of template DNA into short fragments. A universal sequencing primer that included a short DNA sequence unique to the sample (MID tag) was then annealed to both ends of each DNA fragment. A GS Junior Titanium Emulsion PCR Kit (Roche, Mississauga, ON) was used to amplify the sample library, which was sequenced using a GS Junior Titanium Sequencing Kit (Roche, Mississauga, ON). In order to assemble a full genomic sequence, the short sequences produced by 454 sequencing were aligned with the reference FV3 genome, wt-FV3, using GS Reference Mapper (Roche, Mississauga, ON). Any gaps in the assembled genome were then sequenced using custom PCR primers specific to each gap, with sequencing performed by the Robarts Sequencing Facility (London, ON). The final genomic sequence was deposited in GenBank accession number KJ175144.

Morrison E.A., Garner S., Echaubard P., Lesbarrères D., Kyle C.J, & Brunetti C.R. (2014). Complete genome analysis of a frog virus 3 (FV3) isolate and sequence comparison with isolates of differing levels of virulence. Virology Journal, 11, 46.

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16S rRNA Gene Amplification and Sequencing

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The V4 hypervariable region of the 16S ribosomal RNA gene was amplified using GS FLX 454 one way read barcoded fusion primers (Lib-L kit, Primer A, Primer B, Roche 454 Life Science, Branford, CT, USA) with the template specific sequences F515 and R806 (Caporaso et al., 2011a) . PCR triplicates were run using illustra Hot Start Mix RTG beads (GE Healthcare, Little Chalfont, Bucks, UK) following the manufacturer's protocol with 0.4µM of each primer and 1µL of DNA extract. The PCR triplicates were pooled and purified with the QiaQuick Gel Extraction kit (Qiagen, Venlo, Netherlands). After DNA quantification with QuantiFluor dsDNA Dye (Promega, Madison, WI, USA) the samples were pooled equimolar and were run on a 2100 Bioanalyzer (Agilent, Waldbronn, Germany) for quality assessment and quantification. Emulsion PCR of pooled samples was performed with the GS Junior Titanium emPCR Kit (Lib-L) (Roche 454 Life Science, Branford, CT, USA) as per the manufacturer's instructions. Sequencing was run on a GS Junior instrument using GS Junior Titanium Sequencing Kit and PicoTiterPlate Kit (Roche Diagnostics, Basel, Switzerland).

Wedin M., Maier S., Fernandez-Brime S., Cronholm B., Westberg M, & Grube M. (2016). Microbiome change by symbiotic invasion in lichens. Environmental microbiology, 18(5).

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