2G Analyser / Illumina

SOLiD 2 system / Applied Biosystems

454 (FLX Titanium Serie) / Roche

Comparison of Illumina and SOLiD systems

Which system is better? It is impossible to answer question in this form. At the moment both systems have their own advantages and disadvantages. To make a decision it is necessary to think about main applications of the system.

• More or less clear situations:
  • high throughput resequencing of large genomes: SOLiD is more accurate;
  • RNA structure analysis: short-insertion MP-sequencing is available only for Illumina.




parameter Illumina SOLiD   main   throughput ≈   read length ≈   quality ++ +++   coverage uniformity +++ ++   perspectives ≈   price library prep. ≈ flowcell prep. & sequencing ≈   current   procedures fragment libraries ≈ MP-libraries <600bp +++ impossible on beads MP-libraries >600bp ≈ flowcell preparation ++ - sequencing +++ + bioinformatics ≈   in house   technology   development libraries ++ - flowcell preparation - ++ sequencing ++ + data processing ≈   organization kits and consumables +++ - additional equipment ≈ ordering and delivery - +++ stability of the system ++ + service and support + +++






General comparison: Illumina ≈ SOLiD

Let suppose, that it is necessary to resequence human genome with 10x coverage with mate-paired procedure. It is about 10 x 3Gb = 30Gb of mappable nucleotides.

Illumina
Short-insertion MP-library
One MP-run on Illumina gives ~8Gb of raw nucleotides or 7Gb (~88%) of mappable nucleotides.
Number of runs is 30Gb / 7Gb = 4.3 runs. The result is ~4.3x10⁸ of 2x36b reads with error rate ~180 errors per 100 of 2x36bp reads.


Prices:
• library preparation:
       *  $320 (kit) + ~$60 other expenses = $400 on kit reagents;
       *  ~90€ on home-made reagents;
• flowcell preparation: $4000 per FC;
• sequencing: $2400 per FC;
• consumables: ~$65 per FC;
• total price: ($4000 + $2400 + $65) x 4.3 + $400 = $28200 => ~21800€



Time:
• library preparation: a bit less, than 1 week;
• sequencing: 6 days per run, 26 days total;
• total time: ~1.1 month;




Long-insertion MP-library
One MP-run on Illumina gives ~5.25Gb of mappable nucleotides.
Number of runs is 30Gb / 5.25Gb = 5.7 runs. The result is ~5.3x10⁸ of 2x27b reads with error rate ~130 errors per 100 of 2x27bp reads (fewer errors, because of shorter reads).


Prices:
• library preparation: ~150€ on home-made reagents
• flowcell preparation: $4000 per FC;
• sequencing price per FC is lower, because 27 cycles are done instead of 36: $1800;
• consumables: $65 per FC;
• total price: ($4000 + $1800 + $65) x 5.7 + $150 = $33580 => ~26000€



Time:
• library preparation: a bit more, than 1 week;
• sequencing: ~5 days per run, 28 days total;
• total time: ~1.2 month;






SOLiD
Short-insertion MP-library
---- impossible ----



Long-insertion MP-library
One MP-run on SOLiD gives ~11.7Gb of raw nucleotides or 5.0Gb of mappable nucleotides.
Number of runs is 30Gb / 5Gb = 6 runs. The result is ~6x10⁸ of 2x25b reads with error rate ~6 errors per 100 of 2x25bp reads.


Prices:
• library preparation: ~150€ on home-made reagents
• flowcell preparation: 610€ per FC;
• sequencing: 2920€
• consumables: ~100€ per FC;
• total price: ($610 + $2920 + $100) x 6 + $150 = 21930€



Time:
• library preparation: a bit more, than 1 week;
• sequencing: 10 days per run, two FC simultaneously, 30 days total;
• total time: ~1.3 month;




Conclusions:
• time is practically the same;
• more hand work for SOLiD (but one technical assistant is enough in both cases);
• price (w/o equipment) is practically the same (flowcells are more expensive for Illumina, sequencing — for SOLiD);
• equipment price per project is also quite similar, because run time is a bit shorter for Illumina (26 days // 30 days) and price of instrument is a bit less for SOLiD;
• short-insertion MP-sequencing is impossible on SOLiD platform;
• read length of long-insertion MP-sequencing is restricted to 25bases on SOLiD platform (because of the program);
• error rate is much less for SOLiD. It is important for low-coverage projects. But, coverage variance of the SOLiD is about two times higher than coverage variance for Illumina, which is not good for low-coverage analysis. Besides, high variance of coverage on SOLiD makes analysis of structural variations more difficult. Taken together, combination "accuracy + coverage" is better for SOLiD, but not dramatically.
  
  It is also possible to increase the accuracy of Illumina by eliminating reads with low quality. In this case more runs would be necessary to perform, so the overall price would increase.





Throughput: Illumina ≈ SOLiD

• throughput (mappable nucleotides per day) is about the same for both systems: ~1.2 Gb/day. The equivalence is regularly disturbed after upgrading of one of the systems, but upgrading plans for both platforms are quite similar in the nearest future.





Read length (RL): Illumina ≈ SOLiD

• about the same now: ~35b as a kit, ~50b as a home-made modification of protocol. Near-future plans are quite similar.
• increasing of RL have both positive and negative consequences. Optimal RL is different for different applications.





Quality: Illumina < SOLiD

• Accuracy of SOLiD platform is higher, because each nucleotide is analysed twice in ligation-based sequencing. As a result single-nucleotide substitution change colour of two dinucleotides. Standing alone colour changes may be filtered out as sequencing errors.
• Comparison of error frequencies: "standing alone colour mistakes SOLiD" > "Illumina errors" >> "filtered two-dinucleotide colour changes SOLiD".





Perspectives: Illumina ≈ SOLiD

• development plans for near future are quite similar for both platforms: increase of seq-elements density, better chemistry/enzymes, acceleration of imaging, etc.
• long-term development is also similar. From technical point of view, both machines are combination of liquid handling robot with fluorescent scanner. If it would happen, that some particular sequencing approach is much better, than competitors, both robots might be adapted for this procedure.
• if compare polymerisation and ligation approaches, it is unclear now which is more perspective, but unlikely, that perspectives are the same. In both cases progress will be limited (unlikely, that any of the systems would reach read length and quality of the capillary sequencing). But, basic principles are completely different, so one approach will reach internal limitations early, than other.





Fragment libraries preparation: Illumina ≈ SOLiD

• fragment libraries for both systems are DNA fragments with adaptors attached to both ends. Any procedure/kit developed for one system may be relatively easy adapted for the other.
• the same story for any single-read libraries: fragment genomic libraries, barcodes, microRNA libraries, etc.
• minimal amount of starting material is about the same for both systems. Minor difference: ePCR on beads is more restrictive to the fragment length than bridge amplification. If starting material is a set of heterogenious short DNA fragments (chromatin immunoprecipitation) more fragments will be suitable for Illumina sequencing, than for the SOLiD one.





MP-libraries <600bp: Illumina only

• Illumina have developed a technology for changing orientation of clusters directly on the glass. It gives a possibility to sequence library fragments from both ends. The only restriction is the length of DNA fragment. Everything is fine if fragment length is below ~300bp. Fragments in the range 300-600bp also may be sequenced, but with lower efficiency: cluster sizes are heterogeneous, so cluster density should be lower.
• in case of "large-size MP-libraries" read length is limited by the length of restriction fragments. Sequencing of "small-size MP-libraries" may be done with any desirable read length.
• SOLiD have no instrument for the small-size MP-sequencing:
  • MP-libraries can't be constructed from relatively short (<500bp) fragments, because they can't bend to form a circle;
  • in the very beginning Applied Biosystems claimed, that the ligation-based technology would be able to sequence fragments in both directions. But now it works only in 3'→5' direction;
  • problem would not solve completely, even if Applied Biosystems would develop 5'→3' sequencing or template inversion, because "beads / emulsion PCR" does not work well for fragments >200bp





MP-libraries >600bp: Illumina ≈ SOLiD

• large-size MP-libraries differ only by sequence of adaptors. Any protocol may be adapted for both systems. For example, MP-library construction protocol for Illumina presented on this site is based on SOLiD procedure.





Flowcell preparation: Illumina >> SOLiD

parameter Illumina SOLiD comments time +++ - for Illumina is about 3 times less hand-work +++ - much more for SOLiD small-scale library prep +++ -- Illumina: no restrictions SOLiD: it is impossible to prepare PCR emulsion for less than 1/2 of the slide (because of geometry of ULTRA TURRAX DT-20 tube) loading of several samples ++ - Illumina: 8 independent samples may be loaded without any loss of throughput SOLiD: 4 independent samples — loss of 1/4 of glass area; 8 samples — loss of 1/3 of area contamination safety ++ + much better for Illumina procedure flowcell storage + +++ days for Illumina, months for SOLiD






Sequencing: Illumina > SOLiD

parameter Illumina SOLiD comments reagent preparation +++ + Illumina: ready-to-load kit SOLiD: about 4 hours flowcell installation ≈ about the same hand-work hand-work +++ - Illumina: one reagent loading for one-direction sequencing SOLiD: enzyme and primer plates should be changed each day; reagents should be refilled several times during the run






In house development (libraries): Illumina > SOLiD

• the only restriction for the library development for Illumina: distal parts of adapters should correspond to the primers immobilized on the flowcell. Design of alternative sequencing primers is easy and straightforward.
• a lot of inconveniences with SOLiD. As for Illumina, one of the adaptors should correspong to the primer immobilized on beads. It is impossible to change sequencing primer without "opening" the kit, because a special "blocking" and special "bridge" primers should be used (company neither supply information about structure of this primers, nor takes orders for custom synthesis). Besides, at least 5 different primers should be ordered for one sequencing place.





In house development (flowcell): Illumina < SOLiD

• SOLiD flowcell is a plain glass. It is possible to customize bead deposition, etc. Practically nothing is possible to do inside of Illumina's channels.





In house development (sequencing): Illumina < SOLiD

parameter Illumina SOLiD comments modification of the instrument programm +++ - Illumina: easy and clear both for image collection and liquid handling SOLiD: impossible* fluorescence excitation and detection + ++ Illumina: uses two lasers, excitation wavelength are practically fixed SOLiD: UV lamp, in principle it is possible to change filters, but it is unclare how to program

^* EcoP15I MP-libraries have 27b-long fragments, but it is possible to select only (20, 25, 30, 35, 40,...) cycles in SOLiD instrument control software. Changing of read length from 25 to 27 would be really helpful. We tried to push company to modify the program, but without success. On Illumina it would take ~5min to change the number of cycles.




Kits and consumables (convenience): Illumina > SOLiD

• Illumina kits: some reagents should be dispensed by 2µl pipette, but delivered in 1.7ml tubes: Klenow, Klenow exo(-), primers. Practically no other problems;
• a lot of inconveniences with SOLiD kits:
  • misleading names of reagents:
         *  two completely different reagents have similar names: "Bead Wash Buffer" and "Bind & Wash Buffer";
         *  "Low Salt Binding Buffer" instead of "Binding Buffer";
  • cups without labels:
         *  it is difficult to handle 11-26 tubes with primers without cup-labels;
         *  it is possible to change cups by mistake;
  • labels on some tubes completely hide the content of the tube;
  • some reagents, that should be dispensed by 2µl pipette are delivered in large tubes;
  • a lot of reagents may be premixed in advance;
  • there are only oligonucleotide sets for library preparation, complete kits are not available;
  • quantities of viscous solutions (glycerol, ePCR components) should be expressed in terms of weight (not volume);
  • company did not check, if primer plates might be stored frozen;





Additional equipment: Illumina ≈ SOLiD

• both systems require:
  • additional computing facilities for data analysis and storage;
  • room with climate control (NB, SOLiD produce more heat);
• Illumina:
  • nebulizer is a bad instrument for DNA shearing: some ultrasonic device should be ordered;
  • Hydroshear is obligatory for preparation of large-insertion libraries;
• SOLiD:
  • Xstream repeater;
  • should be enough number of 96-well PCR machines for ePCR;





Ordering and delivery: Illumina << SOLiD

• Illumina: delivery takes at least 3 weeks (Europe);
• SOLiD: normally 1-3 days from order to delivery. Excellent.





Stability of the system: Illumina > SOLiD

• both systems are not completely stable. Sometimes (one per ~20) run fails for unknown reasons. Illumina is a little more stable than SOLiD in this respect.
• Illumina uses lasers, SOLiD — UV lamp. Lasers are much more expensive, but UV-lamp has a much shorter operation life (about 0.5 year). Each UV lamp costs ~$600.





Service and support: Illumina < SOLiD

• SOLiD:
  • people from Applied Biosystems are ready to help in installation and training of technical assistants;
  • reparation is fast, broken part delivered in a next day;