A large genome center s improvements to the Illumina sequencing system

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1 28 Nture Pulishing Group A lrge genome center s improvements to the Illumin sequencing system Michel A Quil, Iwnk Kozrew, Frnces Smith, Aylwyn Sclly, Philip J Stephens, Richrd Durin, Hrold Swerdlow & Dniel J Turner The Wellcome Trust Snger Institute is one of the world s lrgest genome centers, nd sustntil mount of our sequencing is performed with next-genertion mssively prllel sequencing technologies: in June 28 the quntity of purityfiltered sequence dt generted y our Genome Anlyzer (Illumin) pltforms reched 1 terse, nd our verge weekly Illumin production output is currently 64 gigses. Here we descrie set of improvements we hve mde to the stndrd Illumin protocols to mke the lirry preprtion more relile in high-throughput environment, to reduce is, tighten insert size distriution nd relily otin high yields of dt. Next-genertion DNA sequencers, such s the 454-FLX (Roche), SOLiD (Applied Biosystems) nd Genome Anlyzer (Illumin) hve trnsformed the lndscpe of genetics through their ility to produce hundreds of megses of sequence informtion in single run. This hs enled us to design genome-wide nd ultr-deep sequencing projects tht, ecuse of their enormity, would not otherwise hve een possile (for reviews see refs. 1, 2 nd for n evlution of the performnce of these three pltforms see ref. 3). At the Wellcome Trust Snger Institute we currently hve ll three of these sequencing pltforms, though the Genome Anlyzer is the pltform we hve invested most hevily in: we hve 28 mchines on site, ll cple of generting pired-end dt. The Illumin dt nlysis pipeline performs purity filtering of these dt to eliminte sequence dt from clusters tht pper to e mixed s consequence of their proximity on the flowcell. We typiclly generte 4 5 gigses (G) of filtered sequence dt, with n error rte of <.9% per seven-dy, 36-cycle pired-end run, mking us one of the world s lrgest nd most productive users of Illumin sequencers. Sequencing lirry preprtion involves the production of rndom collection of dpter-modified DNA frgments, with specific rnge of frgment sizes, which re redy to e sequenced. We hve found the stndrd Illumin sequencing lirry preprtion protocols (Fig. 1) to e suoptiml in severl respects, nd we enhnced our output y developing nd implementing mny modifictions nd improvements to these protocols, ll with the im of otining the mximum numer of high-qulity sequence reds per run from the lowest mss of strting DNA, in roust nd reproducile wy. The modifictions nd improvements we descrie here cn e dopted en msse s n lterntive lirry preprtion pipeline. However, ecuse some steps re dditionl rther thn lterntive, we tend to select different modifictions for different sequencing projects, depending on the specific requirements of tht project (Supplementry Tle 1 online). Here we hve ttempted to descrie ech modifiction in the order in which it would fit in to the stndrd lirry preprtion pipeline. Frgmenttion The first stge in stndrd genomic DNA lirry preprtion for the Genome Anlyzer is DNA frgmenttion y neuliztion (in 3 6% glycerol t 3 35 p.s.i.). This genertes frgments with typicl size rnge of 12 se pirs (p) nd pek round 5 6 p. Neuliztion is firly reproducile technique, is sequence-independent, nd is rpid nd inexpensive 4. However, the wide size distriution of generted frgments is uneconomicl: y mss, the 2 ± 2-p frgments represent only ~1% of the totl DNA fter neuliztion. Moreover, pproximtely hlf of the DNA vporizes during neuliztion, mening tht only 5% of the originl DNA is used for susequent lirry genertion. Even under much more extreme neuliztion conditions (for exmple, 9 p.s.i. for 18 min) it is not possile to move the frgmentsize pek elow round 4 p, nd doing so still does not improve the yield t 2 p (ref. 4 nd unpulished oservtions). Thus we hve evluted lterntive methods of smple frgmenttion. Wellcome Trust Snger Institute, Wellcome Trust Genome Cmpus, Hinxton, Cmridgeshire, CB1 1SA, UK. Correspondence should e ddressed to D.J.T. (djt@snger.c.uk). PUBLISHED ONLINE 25 novemer 28; DOI:1.138/NMETH.127 nture methods VOL.5 NO.12 DECEMBER 28 15

2 Genomic DNA Frgmenttion 1 Frgmented DNA smple ( 12 p) End repir PCR-mplified lirry 12 Denturtion Single-strnded DNA lirry Cycles of cluster mplifiction tocol: (i) is in the se composition of sequences; (ii) high frequency of chimeric sequences, produced when two templte strnds re ligted during the dpter ligtion step; nd (iii) imperfect distriution of insert sizes. These hve ll een overcome y the use of severl protocol modifictions, descried here. 28 Nture Pulishing Group Blunt-ended, 5'- phosphorylted frgments A-tiling Ligtion Size selection 3' A-tiled frgments Adpter-ligted frgments Size-selected frgment lirry PCR PCR-mplified lirry 2 3,4 4,5 6,7,8,9,1 Quntifiction Clonl DNA clusters on flowcell surfce Single-strnded clusters redy for sequencing Incorportion of fluorescent reversile termintor nucleotides Clevge of fluorophores nd locking groups Adptive focused coustics. We now routinely frgment DNA smples using dptive focused coustics technology in 24-well formt (AFA; Covris). In this process, coustic energy is controllly focused into the queous DNA smple y dish-shped trnsducer, resulting in cvittion events within the smple. The collpse of ules in the suspension cretes multiple, intense, loclized jets of wter, which disrupt the DNA molecules in reproducile nd predictle wy. After disruption, 2-p frgments comprise 17% of the totl frctionted DNA y mss, ut in contrst to neuliztion, very little DNA is lost during the frgmenttion process, generting four- to fivefold higher yield of the intended frgment size rnge (Supplementry Protocol 1 online nd Fig. 2). Additionlly, ecuse of its high-throughput cpility, AFA hs enled highly multiplexed sequencing using indexing tgs, where ech smple needs to e processed seprtely until fter PCR mplifiction (Fig. 1). Also, ecuse the size distriution of DNA frgmented y AFA is nrrow, for some pplictions, such s rry enrichment of trgeted loci 5,6, we re le to omit the gel electrophoresis sed size selection step ltogether from the lirry preprtion, decresing the worklod nd incresing yields further. A-tiling, ligtion nd size selection After close scrutiny of pired-end reds otined from the Genome Anlyzer, tht is, those in which ech cluster ws sequenced in oth forwrd nd reverse directions, we discovered numer of rtifcts tht could e ttriuted to the stndrd lirry preprtion pro Lineriztion, locking nd hyridiztion Flowcell trnsfer to Genome Anlyzer Imging Lirry preprtion Cluster growth Sequencing y synthesis Figure 1 Illumin sequencing workflow. Stges in the lirry preprtion. Steps ccompnied y numers re those for which we suggest lterntives to the stndrd Illumin protocols. Numers correspond to those given in Supplementry Protocols 1 13 online. Pired-end oligonucleotides. We no longer use the Illumin single-end dpters or PCR primers ecuse pired-end oligos generte sequencing lirries tht re comptile with oth single nd pired-end flowcells. The dpters themselves re modified to confer protection from digestion t the 3 thymine (T) overhng. Though we do not hve the detils of the modifiction used y Illumin, we hve otined comprle results using our own dpters nd PCR primers modified with phosphorothiote etween the two ses t the 3 end (Supplementry Protocol 2 online). Gel extrction. During the size selection step of the stndrd lirry prep protocol, gel slice is selected nd the DNA extrcted. We identified tht melting this gel slice y heting to 5 C in chotropic uffer decresed the representtion of A+T-rich sequences, possily reflecting higher ffinity of spin columns for doule-strnded DNA, s strnds with high A+T content will e most likely to ecome dentured during this step nd my not rennel. To improve the representtion of these A+T-rich sequences, we modified the gelextrction protocol, melting grose gel slices in the supplied uffer t room temperture (18 22 C). This reduces G+C is considerly (Supplementry Protocol 3 online nd Fig. 3,). Doule size selection. Prtilly complementry dpters, which essentilly consist of the sequences to which the sequence primers hyridize during the sequencing rection, re ligted onto the A-tiled frgments 7 vi T overhng (Fig. 1). Their structure ensures tht ech templte strnd receives different sequences t the opposite ends 8 nd works in much the sme wy s vectorette 9. Inefficient end-repir or A-tiling rections will result in lower concentrtion of templte to which dpters cn e successfully ligted, nd so the reltive concentrtion of dpters is incresed, which will promote the formtion of dpter dimers. If these dimers re not removed, they will ultimtely e sequenced long with the intended templte, wsting the cpcity of the flowcell. Additionlly, inefficient A-tiling will result in high proportion of lunt-ended templte molecules, which cn self-ligte, generting chimeric sequences. It is likely tht the efficiency of the A-tiling step will e improved y the use of lterntive polymerses nd higher concentrtions of mgnesium ions. Additionlly, the efficiency of the ligtion step ppers to e improved y the use of ultrpure ligses, such s those from Enzymtics, which re virtully free of the contminting exonuclese ctivity generlly found in stndrd commercil 16 VOL.5 NO.12 DECEMBER 28 nture methods

3 28 Nture Pulishing Group Reltive fluorescence units ,5 preprtions of ligses. Using this enzyme we hve chieved 2 3% increse in yield of successfully ligted frgments (s determined y quntittive PCR (qpcr); Supplementry Protocol 4 online), presumly ecuse the reduced exonuclese ctivity regenertes fewer lunt-ended frgments fter A-tiling. However, lthough the steps descried ove my reduce the formtion of luntended ligtion conctmers, enzymtic rections re rrely 1% efficient, nd thus smll proportion of templte strnds will still e chimeric. In mny pplictions, low frequency of chimeric sequences will present no prolem nd cn simply e removed informticlly. In other pplictions, such s detection of infrequent de novo recominnt molecules, chimeric sequences will generte flse positives, so their frequency needs to e reduced to minimize the mount of susequent confirmtory work. Chimeric templtes will e longer thn the singletons, which provides wy of preventing them from contminting the DNA frction: for pplictions in which low frequency of chimeric templtes is required, we perform n dditionl size selection fter shering ut efore ligtion (Supplementry Protocol 5 online). This results in nrrow size rnge eing ville for ligtion. Any lunt-ended conctemers re pprecily longer thn the singletons, nd we remove these in the postligtion size-selection step. This dditionl size selection reduces the incidence of chimers to.2%, compred to up to 5% with the stndrd lirry preprtion protocol, nd we hve found this step to hve the dded enefit of reducing the shoulder of smll insert sizes, giving tighter insert-size distriution of the desired frction (Fig. 3c e), which leds to clusters with more uniform dimeter. Reltive fluorescence units Mpped depth (in size, 5 p) Neuliztion AFA Ldder Figure 2 Smple frgmenttion. Comprison of frgmenttion y neuliztion with AFA technology. We frgmented 4.5 µg of humn genomic DNA y neuliztion or AFA, purified the smples using spin column, eluted the DNA in 3 µl of 1 mm Tris ph 8.5, nd rn 1 µl of ech elute on n Agilent Bionlyzer 21 DNA 1 chip. For 2-p (±2 p) lirry, the yield produced y AFA ws four- to fivefold greter thn tht produced y neuliztion. Numer of reds (millions) G+C content (%) Percentile of unique sequence ordered y G+C content , Pired-end size selection. Using stndrd protocols, we found singleend lirry preprtions, using single- or pired-end dpters, to e considerly more roust t the step of size selection y electrophoresis thn their pired-end counterprts. With single-end preps, we excise nd of 5 p or lrger, which generlly yields more thn enough DNA to give high yield of PCR products. However, for the pired-end protocol, to generte s nrrow n insert size rnge s possile, sclpel is inserted into the gel t the desired position, the lde is wshed with Tris uffer, nd this uffer cts s the templte for the PCR mplifiction. We found this prctice to yield enough DNA to give successful mplifiction only in pproximtely 3 4% of ttempts. To overcome this, we now excise 2-mm-wide gel slice contining DNA of the desired size nd extrct tht following the Illumin protocol, though with no heting during the melting step, s discussed ove (Supplementry Protocol 3). In our hnds this typiclly yields 1 2 times more DNA thn the stndrd protocol, hs n lmost 1% success rte nd genertes n cceptly nrrow size distriution of pired-end reds (Fig. 3f,g). PCR As well s incresing roustness, extrcting more DNA from gel slices enles the DNA to e quntified more ccurtely efore PCR mplifiction. The PCR step introduces into the dpter-ligted templte molecules the oligonucleotide sequences required for hyridiztion to the flowcell surfce. Reltive fluorescence units Distriution of reds with indicted G+C content Men red depth S.d. c d e f g Numer of reds (millions) Mpped depth (in size, 5 p) , G+C content (%) Percentile of unique sequence ordered y G+C content Reltive fluorescence units Distriution of reds with indicted G+C content Men red depth S.d ,5 Figure 3 A-tiling, ligtion nd size selection. (,) G+C plots efore () nd fter () optimiztion of gel extrction. The figures show the totl re in which reds with prticulr G+C content re distriuted, with the men nd s.d. The greter width of the shded re in plot indictes wider dispersion of coverge for ll vlues of G+C content for which sequences were otined. (c e) Agilent Bionlyzer 21 trces for three lirries, 6-p insert lirry with optimized PCR (c), the sme 6-p lirry with excess DNA in PCR (d) nd 2p insert lirry, showing shoulder of smll frgments (e). (f,g) Insert size distriution from sequenced humn DNA using the stndrd (f) nd modified (g) pired-end lirry preprtion protocols. nture methods VOL.5 NO.12 DECEMBER 28 17

4 Optiml PCR conditions Stndrd PCR conditions Figure 4 PCR. () An ~2-p frgment lirry ws prepred, nd 1 ng ws mplified for 18 cycles using stndrd Illumin PCR conditions or optimized PCR conditions. () A comprison of methods of PCR mplicon purifiction. We prepred pired-end lirry with phix DNA using conditions tht would promote the formtion of dpter dimers nd unextended PCR primers. After PCR, we divided the lirry into two: hlf ws purified following the stndrd Illumin protocol, through QiQuick PCR clenup column (left), wheres the other ws purified using SPRI technology (right). 28 Nture Pulishing Group Adpter dimers PCR primers ,5 Column clenup Ldder Bnd size (p) 1,4 1, 1, Ldder SPRI clenup Templte quntity. By using optimized quntities of templte in the PCR, we cn ensure clen lirry, free of dpter-dimer or singlestrnded DNA. We routinely nlyze our sequencing lirries fter PCR y microfluidic cpillry electrophoresis nd hve noticed tht the qulity of the lirry otined decreses with incresing concentrtion of templte DNA: too much templte DNA often results in the ccumultion of n pprently higher-moleculr-weight pek (Fig. 3d), which my represent single-strnded templte product tht ccumultes s primers ecome depleted. Conversely, the lower the mss of DNA used in the PCR, the fewer the numer of templte strnds for the sme frgment size, nd the greter the incidence of PCR product duplictes in the resulting sequences: we hve oserved lirries from which s mny s 6% of sequences were PCR product duplictes. Thus it is essentil to choose the pproprite set of conditions for ech PCR (Supplementry Protocol 6 online). PCR yield. By the use of lterntive high-fidelity polymerses in more optimized rection, we hve found it possile to increse the yield of the enrichment PCR five- to tenfold (Supplementry Protocol 7 online nd Fig. 4), which llows fewer cycles of mplifiction to e performed. PCR clenup. Surplus PCR primers my interfere with quntifiction nd will compete with the mplicon for hyridiztion to the flowcell surfce. Consequently, it is necessry to remove surplus oligos fter mplifiction. We hve found tht solid-phse reversile immoiliztion (SPRI) technology 1 cn e used to remove higher proportion of primers nd dpter dimers thn spin columns, while producing comprle yield of mplicon DNA, nd llows elution in wider vriety of uffers (Supplementry Protocol 8 online nd Fig. 4). Sequencing without PCR. We hve found tht it is unnecessry to retin the PCR step to enrich for properly ligted frgments, so long s only those frgments with n dpter t either end cn e quntified, s only they will yield clusters tht cn e sequenced. This cn e done y quntittive PCR, discussed elow. Thus we cn eliminte the PCR step entirely, simply y ligting on pproprite dpters fter A-tiling (Supplementry Protocol 9 online). For this purpose we use high-performnce liquid chromtogrphy purified, prtilly noncomplementry oligos with phosphorothiote linkge etween the two ses t the 3 end of one strnd. From strting mount of 5 µg of DNA nd frctiontion y AFA, we cn otin sufficient pired-end DNA for > 4 lnes of high-density clusters, or 1 lnes if neuliztion is used to frgment the DNA. The ovious enefits of this re tht PCR duplictes re sent: the oserved dupliction Numer of clusers fter purity filtering 35, 3, 5-p frgments 25, 2-p frgments 2, 15, 1, 5, 1, 2, 3, 4, 5, 6, 7, Totl numer of clusters detected Unfiltered cluster numer per tile 7, 5, 3, 1, Run numer Medin cluster numer Upper nd lower qurtiles 1.5 interqurtile rnge Outlier Fluctution in medin cluster numer from in to in qpcr ssy introduced Figure 5 Quntifiction. () Cluster throughput s function of totl clusters for 2- nd 5-p inserts. The 5-p inserts underwent fewer cycles of cluster mplifiction (28, compred to 35 for the 2-p lirries), resulting in smller clusters, nd so cluster density of 4 44k per tile (GA1) will produce the mximum yield from either insert size. () Stndrdiztion of cluster density with qpcr quntifiction. Runs were grouped into 25-run ins, nd oxplot ws generted. After some initil prolems with degrdtion of stndrds, cluster numer hs leveled out t ~35, 4, per tile. 18 VOL.5 NO.12 DECEMBER 28 nture methods

5 28 Nture Pulishing Group Modified hyridiztion uffers. For ll denturtion we prefer the use of.1 M NOH to heting, though for sunnomolr lirries this requires n lterntive hyridiztion uffer to e used (Supplementry Protocol 12 online). We hve found the ddirte in mpped gorill DNA sequences prepred without PCR ws pproximtely.5%. This rte of dupliction is cused y noise in the cluster detection nd sequence nlysis softwre. Direct sequencing of short mplicons. To void unnecessry PCR mplifiction steps, which would potentilly excerte ises, we cn perform extremely deep sequencing of short mplicons using locusspecific primers tht possess tils tht cn hyridize to the oligos tethered to the flowcell surfce. The tilless forwrd nd reverse oligos re then used s primers in the sequencing steps (Supplementry Protocol 1 online). Quntifiction At close to neutrl ph, the concentrtion of DNA going onto the flowcell governs the numer of clusters produced. Thus, for different frgment sizes undergoing given numer of cycles of cluster mplifiction, there is n optiml concentrtion rnge of DNA tht will yield clusters in the optiml density rnge, enling the mximum mount of dt to e otined. For frgments with men insert size of 5 p or lower, we im for 4, 44, clusters per imged re (tile) on the Genome Anlyzer model 1, giving n verge of 2, 25, filtered clusters per tile, equting to G per single end run (15 17, clusters per tile for the Genome Anlyzer model 2; Fig. 5). Overestimtion of DNA concentrtion results in too few clusters, which my mke the flowcell uneconomicl to sequence. Underestimtion results in too high cluster density, which cn gretly reduce the mount of dt otined, owing to cluster overlp. Quntifiction of DNA efore sequencing is thus one of the key fctors in the process. Electrophoresis. We found the ccurcy of spectrophotometry to e indequte for quntifiction: cluster density sed on this method tended to e inconsistent, ut typiclly five- to tenfold lower thn nticipted, presumly ecuse spectrophotometry nlysis mesures not only the intended mplicon ut lso dpter dimers nd unextended primers, with no wy of distinguishing etween them, nd lso struggles to mesure low DNA concentrtions ccurtely. By quntifying lirries electrophoreticlly, with n Agilent Bionlyzer, we hve een le to chieve much more consistent cluster density. Additionlly, ecuse electrophoresis cn e used to distinguish etween DNA species on the sis of size, it provides wy to check the qulity of the lirry preprtion. However, for smll proportion of lirries, we otined fr higher cluster densities, nd consequently fr less useful dt, thn nticipted. We ssume tht this is result of single-strnded DNA generted in the PCR tht cnnot e esily quntified when mixed with doule-strnded DNA. Quntittive PCR. This led us to develop qpcr quntifiction ssy (for discussion see ref. 11) ecuse such n pproch should e cple of detecting nd quntifying ll mplifile molecules. We designed mplifiction primers nd dul-leled proe to trget the Illumin pired-end dpter sequences (Supplementry Protocol 11 online). We quntify unknown lirries ginst stndrd lirries tht hve een sequenced previously, nd for which we know the ccurte cluster numer, nd how this reltes to the Agilent concentrtion of tht lirry. Becuse mplifiction in the qpcr with these primers is not perfectly efficient, we use 3 dilutions of stndrd lirries (1, 1 nd 1 pm), nd dilute the unknown lirry to 1 pm, sed on the concentrtion s mesured using n Agilent Bionlyzer 21. With this ssy, we tke the Agilentderived concentrtion vlues to e ritrry, llowing us to dilute unknowns to concentrtion tht lies within the 1 1 pm rnge, ut Agilent vlues lso provide useful doule-check. We hve found tht cluster density cn e predicted relily in this wy (Fig. 5). We hve found tht the ility to quntify DNA in the picomolr concentrtion rnge lso opens up the opportunity for sequencing much lower DNA concentrtions thn those permitted y the stndrd protocol, such s unmplified rry elutes from sequencecpture experiment 5,6. Denturtion Being single-strnded, rry elutes require no prticulr steps to denture the DNA 12 efore sequencing. However, for low concentrtions (<1 nm) of doule-strnded DNA it is more prolemtic: denturtion y heting hs the potentil oth to dmge the DNA nd to introduce nti-(g+c) is 13. Cluster/tile ph , 2, 1, Volume.1 M NOH dded (µl) Volume dentured templte dded (µl) Wter 5 SSC 5 SSC + 2 mm Tris 5 SSC + 1 mm Tris 5 SSC + 5 mm Tris 5 SSC 5 SSC + 5 mm Tris Figure 6 Denturtion. () ph titrtion of hyridiztion uffers. Following denturtion, the concentrtion of NOH in DNA templtes is.1 M NOH. Adding more thn 8 µl of this dentured templte to the 1 ml of Hyridiztion Buffer (5 SSC,.1% Tween-2), efore loding DNA onto the flowcell, increses the ph to ove 1. This prevents efficient hyridiztion, nd thus the cluster density flls. The ddition of Tris-HCl ph 7.3 to the supplied ottles of Hyridiztion Buffer drmticlly increses uffering cpcity, mking templte hyridiztion more roust. () The ddition of 5 mm Tris-HCl ph 7.3 to Illumin Hyridiztion Buffer llows greter volume of dentured templte to e dded efore high ph prevents effective nneling of templtes to the oligos on the flowcell surfce. This increses the roustness of cluster genertion y countercting pipetting errors in the denturtion step. nture methods VOL.5 NO.12 DECEMBER 28 19

6 28 Nture Pulishing Group perspective tion of Tris to the stndrd Illumin Hyridiztion Buffer to e eneficil to the roustness of the initil hyridiztion of DNA to the flowcell for ll lirries, ecuse it cn counterct pipetting errors during the denturtion stge tht would otherwise rise the ph to level tht would prevent efficient hyridiztion (Fig. 6). Additionlly, diluting the supplied 2 M NOH nd dding greter volume to the 2 µl denturtion rection helps to reduce fluctution in cluster numer owing to pipetting errors (Supplementry Protocol 12). Amplifiction qulity control After cluster mplifiction, DNA on the flowcell is doule-strnded, which llows clusters to e stined y n interclting dye nd to e detected using fluorescence microscope (Supplementry Protocol 13 online). This is vlule qulity control step, tht we use for ll flowcells efore lineriztion nd locking to confirm tht the cluster density is pproprite. We generlly do not sequence dt from flowcells tht hve too high or too low cluster density eyond the mplifiction stge. Conclusion The Genome Anlyzer is powerful sequencing technology, yet still reltively new, nd consequently it hs not yet reched its full sequencing potentil. Here we hve descried modifictions tht llow for more efficient lirry preprtion nd enle stle workflow in production environment. At the Snger Institute, in ddition to sequencing reserch nd development tem we hve severl tems who re responsile for keeping the production instruments running. A lirry-mking group processes smples, nd genertes, qulity-controls nd quntifies lirries. A production group, working in shifts, prepres nd qulity-controls flowcells y SyrGreen stining, prepres regents for sequencing nd mnges wshing, priming nd loding the instruments seven dys per week. Informtics tems re responsile for fcilitting smple trcking, for hndling the sequence dt nd for performing pipeline nlyses. All steps in the process re recorded using custom-written l-trcking nd run-trcking dtse softwre. All Genome Anlyzers re networked, nd the generted imge dt re continully uploded to lrge compute nd disk-storge cluster for imge nd se-clling nlysis, lignment nd ssemly, nd other informtics tsks. We keep imges for out 1 month on disk server, ut we store the run qulity control nd other run detils in dtse nd deposit short-red sequences for permnent storge in lrge repository. A tem of project mngers coordinte nd oversee individul sequencing projects. We hve recently upgrded ll of our Genome Anlyzers to the model 2. The wider flowcells used y upgrded mchines offer 4% greter imging re, with the potentil for incresed red lengths (>7 ses) of higher qulity (elow 1% error in phix control lne for 1 5 ses). Comined with improvements to the imge nlysis softwre nd fster run time, oth of which we re currently testing, conservtive prediction is tht y the end of 28, our output will rech 6 1 terses of high-qulity sequence per yer, equivlent to 18 humn genomes t 15-fold coverge, or pproximtely 2, ses per second. The improved workflow nd high yield should mintin the Genome Anlyzer s our next-genertion sequencing pltform of choice for the immedite future. How long this remins true depends upon the performnce of existing rivl technologies: Roche s 454, ABI s SOLiD, Helicos True Single Molecule Sequencing nd Dover Systems Polontor, nd those tht re on the horizon, such s nnopore technologies, for exmple Oxford Nnopore Technologies, the Hrvrd Nnopore Group, nd Pcific Biosciences Single Molecule Rel Time technology, which promise to ring us closer to the egerly nticipted $1, genome. Note: Supplementry informtion is ville on the Nture Methods wesite. ACKNOWLEDGMENTS We thnk ll the stff t Illumin for their support, prticulrly T. Ost, M. Gis, J. Smith, N. Gormley, V. Smith nd K. Hll. We lso thnk C. Brown, A. Brown, R. Pettett, T. Skelly, N. Whiteford, L. Mmnov, E. Sheridn nd E. Huckle for helpful discussions nd ssistnce. COMPETING INTERESTS STATEMENT The uthors declre competing finncil interests: detils ccompny the full-text HTML version of the pper t Pulished online t Reprints nd permissions informtion is ville online t nture.com/reprintsndpermissions/ 1. Bentley, D.R. Whole-genome re-sequencing. Curr. Opin. Genet. Dev. 16, (26). 2. Mrdis, E.R. The impct of next-genertion sequencing technology on genetics. Trends Genet. 24, (28). 3. Smith, D.R. et l. Rpid whole-genome muttionl profiling using nextgenertion sequencing technologies. Genome Res. 18, (28). 4. Surzycki, S. DNA sequencing. in Bsic Techniques in Moleculr Biology (Springer-Verlg, Berlin, 2). 5. Alert, T.J. et l. Direct selection of humn genomic loci y microrry hyridiztion. 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