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1 GenePool Information Sheet #1 Installed Sequencing Technologies in the GenePool The GenePool offers sequencing service on three platforms: Sanger (dideoxy) sequencing on ABI 3730 instruments Illumina SOLEXA short read sequencing on the Illumina GAII platform Roche 454 pyrosequencing on the Roche 454 Titanium platform. Platform ABI 3730 Illumina GAII Roche 454 Titanium Specifications and applications DNA sequencing from PCR products, plasmids and other templates up to 800 bases. We offer services from sequence analysis only (we run your completed reactions) to full shotgun analysis of large insert clones (including construction of shotgun libraries and assembly). Samples may be submitted individually or in 96 well plates. We also perform analysis of fluorescently labeled genotyping experiments (microsatellites or AFLP). The instrument can generate up to 8 million reads per lane, and there are 8 lanes per run. Sequences can be from 18 bases to 50 bases (and soon longer) Sequences can be generated from both ends of inserts (paired-end sequencing, 50 bases from each end). The instrument can generate over 5 Gbase per week. Applications include de novo genome sequencing genome resequencing and targeted genome resequencing RNA-Seq (transcriptome sequencing) deep SAGE (digital transcriptomics) smallrna sequencing (microrna sequencing) ChIP-Seq (chromatin immunoprecipitation sequencing) RNA-IP_Seq (RNA immunoprecipitation sequencing) and variants thereof. The instrument can generate up to reads per run Sequence lengths are up to 450 bases, and can be generated from both ends of inserts (paired-end sequencing, 200 bases from each end). Each run can be split into up to 16 lanes. Each run can generate up to 400 Mbase of sequence. Applications include de novo genome sequencing genome resequencing and targeted genome resequencing transcriptome sequencing mass sequencing of PCR products for (e.g.) biodiversity surveys and variants thereof.

2 GenePool Information Sheet #7 June 2009 Comparison of Installed Sequencing Technologies in the GenePool This matrix illustrates the different outputs of the sequencing technologies available at the GenePool, and also indicates the kinds of research applications to which they are suited. The global activity in next generation sequencing is such that new applications are being devised frequently. The GenePool strives to offer these as they mature. Features: Platform: ABI3730 (Sanger dideoxy) Roche Titanium (454) Illumina GAII (SOLEXA) Data per instrument run (single-end) 0.5 Mb 400 Mb 5000 Mb Reads per instrument run (single-end) ,000 >70,000,000 Length of reads up to 900 bases up to 500 bases up to 75 bases Typical cost, per base of raw data Time for one instrument run 4 hr 12 hr hr Can do part runs: up to 16 lanes up to 8 lanes Paired-end sequencing (length of read pairs) (600 b) (200 b) (50-75 b) Applications De novo Genome sequencing: () Genome resequencing De novo Transcriptome sequencing (from clones) Global transcript abundance measurement (deep SAGE and RNA-Seq) Chromatin immunoprecipitation (ChIP) and RNA-precipitation (RNAIP) sequencing Small RNA (including mirna) sequencing Population genetics, phylogenetics and DNA barcoding from individual specimens Population genetics, phylogenetics and DNA barcoding from communities End-sequencing of large-insert clones. Genome or large-insert clone de novo sequencing () Genome or large-insert clone resequencing Metagenomic sequencing

3 GenePool Information Sheet #2 Sanger (dideoxy) sequencing and genotyping in the GenePool The GenePool offers sequencing service on ABI 3730 instruments to users in Edinburgh and in NERC area science*. We run two ABI 3730, 48-capillary sequencers, and are able to process ~ten 96 well plates per day. In 2008 we processed over 200,000 samples, including 170,000 Sanger sequencing reactions and 46,000 genotyping lanes. Sanger sequencing We provide a range of services from complete (where we design and carry out the experiment) to just running your sequence reactions on our instrument. For just electrophoresis samples, turnaround is usually 24 hours. You can also provide us with DNA template (either PCR product or plasmid) and we will carry out the sequencing reaction and analysis. These services are available whether you have one sample or thousands, but are cheaper for large projects if you supply samples in 96-well microtitre plates. We also sequence from recombinant bacterial clones, where you supply the plasmids to be sequenced in the form of 96-well plates containing bacterial cultures. We will PCR amplify the insert and generate sequence from one or both ends. We also offer a shotgun sequencing service, where we will shotgun subclone from your large-insert bacterial clone (fosmid or BAC) and sequence to a specified coverage (usually 6-8 fold). We then perform a draft assembly of your clone and can perform finishing sequencing reactions if required. Genotyping We are happy to provide a genotyping service for existing marker sets (including multiplexed marker sets) of microsatellite and other genetic markers. The minimum number of samples analysed is 48. The ABI 3730 is compatible with the standard range of fluorescent markers. We also offer bioinformatics support to users of the Sanger service for processing of large numbers of samples, etc. We do not usually offer Sanger sequencing service outside Edinburgh, as we do not intend to compete with the local Sanger sequencing services available in most institutions. Contact us to enquire if you do not have access to a local sequencing facility. * Access to the GenePool for NERC science users is managed by the NERC Biomolecular Analysis Facility - see conditions and methods for application for service at

4 GenePool Information Sheet #3 Illumina SOLEXA sequencing in the GenePool The GenePool offers sequencing service on the Illumina GAII instrument to users in Edinburgh, the SULSA universities and in NERC area science*. The technology The Illumina GAII uses SOLEXA short read sequencing technology. This sequencing method relies on two innovations. The first is in vitro cloning, the amplification of single target DNA molecules by solid phase PCR on a microfluidics slide or flow cell (see left). The DNA to be sequenced is first sheared to a uniform length ( bases) before being modified by the addition of adapters and processed in the flow cell. This results in clusters of ~1000 DNA molecules about 1 nm across. The GAII uses a density of up to 60 million clusters per run. The second innovation is the use of a set of fluorescently labeled reversible terminating nucleotides. In each sequencing cycle the DNA template is elongated by one nucleotide. This is read after illumination by a laser by a fixed camera, and the base-addition cycle is then repeated after deblocking and removal of the fluorophore from the terminal base. The sequence for any one cluster is determined by first aligning the individual sets of four images (one for each base see below) from each cycle and then reading off the sequence. The GenePool has good experience in running up to 50 base sequencing reads with very little drop-off in quality even in later cycles. We can also run paired end sequencing, where up to 50 base reads are derived from both ends of each template. Thus each lane of the a GAII flow cell can currently yield up to 375 Mb in a single-end 50 base run, or 600 Mb in paired end 50 base reads. Longer reads and higher read numbers will be available soon. Analysis There exist several high quality and innovative software tools for analysis of short-read sequencer data. The task of turning the images of the clusters into DNA sequence is achieved by on-instrument software, and yields a simple text file of each sequence and the estimated quality of each base called. This quality is derived from the intensity of the fluorescent signal measured, and the level of interfering signal from the other, competing bases. The GenePool bioinformaticians will perform this task for you. Further analysis depends on the kind of data being generated (genome sequencing, RNA sequencing, etc) and can also be performed for you by GenePool staff. The GenePool securely archives all analysed sequence data for at least 1 year after generation. SULSA is the Scottish Universities Life Science Alliance. While SULSA only includes some of Scotland s universities, in practice we offer service to all academic research institutions in Scotland. * Access to the GenePool for NERC science users is managed by the NERC Biomolecular Analysis Facility - see conditions and methods for application for service at

5 GenePool Information Sheet #4 June 2009 Roche 454 Sequencing at the GenePool Sample fragmentation: Genomic DNA and BACs samples are broken into fragments of 300- to 800-bases. Small noncoding 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 singlestranded 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 denovo assembly, resequencing, or variance detection can be performed. 454 Sequencing is a massively parallel, highthroughput pyrosequencing system, delivering up to 0.8 million 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 service from delivery of nucleic acids (genomic DNA or total RNA) to return of assembled data.

6 GenePool Information Sheet #5 Sequencing bacterial genomes with next generation technologies A common requirement of bacterial genetics studies is the de novo determination of the complete sequence of a species or strain of interest 1. Using Sanger technology, the best practice protocols for achieving the complete sequencing of a bacterial genome involve generation of several sequencing libraries with different sized inserts, sequencing the genome to effective several fold depth from these libraries, and then using assembly tools to infer a draft sequence. This draft sequence, often in many fragments, is then refined by additional sequencing to span gaps, before a final assembly is produced, ready for annotation Next generation technology speeds up (and reduces the cost of) the first draft, data generation part of the bacterial genome sequencing process. It is relatively easy to generate many-fold coverage of genomes. Because the next generation technologies do not include a step that involves cloning in bacterial vectors, coverage is usually much more even, and is little affected by different AT/GC proportions in the genome. For de novo sequencing of a bacterial genome, we thus suggest the following (a) sequencing to ~10 fold coverage in 400 base Roche FLX Titanium reads (b) sequencing to at least 20x and preferably 30x coverage from two Illumina GAII libraries (having different mean insert lengths, 250 and >400 bases) using 50 base paired end reads. Assembly of these data with dedicated next-generation assemblers such as Velvet 2 and Newbler 3, will probably yield contigs with well over 98% of the genome in them. However these contigs (there could be as few as 10, and as many as 100 or more) will not be joinedup into a complete genome. The number of contigs that are recovered from an assembly of this kind depend very sensitively on the size of the genome (bigger genomes yield more contigs) and the repeat content of the genome (in the case of bacteria this means the content of IS elements and prophage in the main, although other sequence repeats can also cause breaks in the assembly). The strategy thereafter will depend on your needs for the project. If you want a complete, circular genome, additional sequencing will need to be done to link the contigs. Depending on the number of contigs and the kind of genome, this could involve performing PCR across the sequence gaps, and then directed Sanger sequencing of the PCR products, or generation of a larger-insert plasmid library and end sequencing a low coverage of clones to identify those that span expected gaps. The GenePool is happy to supply quotes for draft sequencing and finishing of bacterial genomes, and for bioinformatic support for subsequent first-pass annotation. 1 Resequencing of bacterial genomes using next generation technology is very much easier than de novo genome sequencing. See the Bacterial Genome Resequencing InfoNote. 2 Zerbino DR, Birney E. Genome Res. 2008;18: Velvet: algorithms for de novo short read assembly using de Bruijn graphs. 3 Newbler is proprietary software of Roche designed for FLX reads.

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