Roche 454 Sequencing at the...
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GenePool Information Sheet #4 v2 July 2009 The Gene Pool (The University of Edinburgh Sequencing Facility) Ashworth Laboratories, King's Buildings, Edinburgh, Scotland EH9 3JT, UK phone 0131 6513633 | email [email protected] | http://genepool.bio.ed.ac.uk/ The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336. Roche 454 Sequencing at the GenePool 454 Sequencing is a massively parallel, high- throughput pyrosequencing system, delivering up to 0.8 million 400-500 base sequences per 12-hour instrument run, using the new GS FLX Titanium series reagents. Paired end sequencing can also be carried out, generating 200 base sequences at each end of inserts up to several kb. The platform can sequence from up to 16 distinct samples at once, and molecular indexing is also possible. Roche 454 sequencing is suited to genome sequencing and resequencing, and de novo transcriptome sequencing in particular. When combined with the massive coverage achievable with the Illumina SOLEXA instrument, whole-genome assemblies of smaller genomes are easily achieved. The 454 can perform: • Transcriptome sequencing • Genome sequencing de novo (including large-insert clone sequencing) • Genome resequencing • Metagenomics sequencing. • Hybridisation-selected DNA sequencing, from Nimblegen or other platforms We offer a complete workflow from delivery of nucleic acids (genomic DNA or total RNA) to return of assembled data. Contact us for more information and a quotation [email protected] Sample fragmentation: Genomic DNA and BACs samples are broken into fragments of 300- to 800-bases. Small non- coding RNA or PCR amplicons, don’t need to be fragmented. Library preparation: Short adapters are added to the 3' and 5' ends of each fragment for purification, amplification, and sequencing. One read per bead: The single- stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Emulsion PCR Amplification: Each unique sample library fragment is amplified within its own microreactor, to exclude competing or contaminating sequences. Pyrosequencing: Amplified fragments are enriched, and sequencing enzymes added. Addition of complementary nucleotides produces a chemiluminescent signal that is recorded by CCD camera. Bioinformatics: Reads are checked for purity, then de- novo assembly, resequencing, or variance detection can be performed. Image used with kind permission of Roche Applied Science
Transcript of Roche 454 Sequencing at the...
GenePool Information Sheet #4 v2 July 2009
The Gene Pool (The University of Edinburgh Sequencing Facility)
Ashworth Laboratories, King's Buildings, Edinburgh, Scotland EH9 3JT, UK
phone 0131 6513633 | email [email protected] | http://genepool.bio.ed.ac.uk/ The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
Roche 454 Sequencing at the GenePool
454 Sequencing is a massively parallel, high-throughput pyrosequencing system, delivering up to 0.8 million 400-500 base sequences per 12-hour instrument run, using the new GS FLX Titanium series reagents. Paired end sequencing can also be carried out, generating 200 base sequences at each end of inserts up to several kb. The platform can sequence from up to 16 distinct samples at once, and molecular indexing is also possible. Roche 454 sequencing is suited to genome sequencing and resequencing, and de novo transcriptome sequencing in particular. When combined with the massive coverage achievable with the Illumina SOLEXA instrument, whole-genome assemblies of smaller genomes are easily achieved. The 454 can perform: • Transcriptome sequencing • Genome sequencing de novo (including large-insert clone sequencing) • Genome resequencing • Metagenomics sequencing. • Hybridisation-selected DNA sequencing, from Nimblegen or other platforms We offer a complete workflow from delivery of nucleic acids (genomic DNA or total RNA) to return of assembled data.
Contact us for more information and a quotation [email protected]
Sample fragmentation: Genomic DNA and BACs samples are broken into fragments of 300- to 800-bases. Small non-coding RNA or PCR amplicons, don’t need to be fragmented. Library preparation: Short adapters are added to the 3' and 5' ends of each fragment for purification, amplification, and sequencing. One read per bead: The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Emulsion PCR Amplification: Each unique sample library fragment is amplified within its own microreactor, to exclude competing or contaminating sequences.
Pyrosequencing: Amplified fragments are enriched, and sequencing enzymes added. Addition of complementary nucleotides produces a chemiluminescent signal that is recorded by CCD camera. Bioinformatics: Reads are checked for purity, then de-novo assembly, resequencing, or variance detection can be performed. Image used with kind permission of Roche Applied Science
