Sequencing by ligation

[MDiamo]

Can someone tell me what are the advantages/disadvantages of sequencing by ligation versus sequencing by synthesis? Is read length restricted in SOLiD because of sequencing by ligation?


Thanks

[pmiguel]

Can someone tell me what are the advantages/disadvantages of sequencing by ligation versus sequencing by synthesis? Is read length restricted in SOLiD because of sequencing by ligation?

Advantages:
(1) Multiple base encoding methods become possible, although currently only dual base encoding is used, and only by AB/Lifetech. The advantage here, in theory, is that much higher accuracy is possible using multiple base encoding schemes. Each base is interrogated twice. The chance of both calls being wrong is the square of the chance of either being wrong. If the factors causing the miscalls are independent...

(2) Ligases are direction agnostic. Polymerases only create nascent strands in a 5' -> 3' direction. The other ligase-using instrument, the Polonator, uses both types of ligations in their protocols. It creates a kind of "paired end" read from a single template. Hilariously, the Solexa, using a polymerase based sequencing system, has had "paired end" read technology almost from the beginning. Whereas, the SOLiD does not yet have this technology. (Looks like it will soon, however. I think it will be part of the v4 upgrade next month.)

Disadvantages
(1) Dual base encoded data looks strange to the naive user. It creates an additional barrier to entry where none exists with single base encoded data.
(2) The SOLiD is slower (per base added) than the Solexa. The reasons for this are complex. But using a ligase instead of a polymerase does play a role, I think, if for no other reason than a lot more work has been done over the years improving polymerase functioning for sequencing. Ligases have not really been optimized for this purpose.

Many of the methodologies utilized by both SOLiD and Solexa derive from work from in the Church lab, published in Shendure et al, [1].

In practice, though it seems Illumina and AB have commercial and legal death grips on each others throats, the uses to which their respective instruments are used may be diverging. The Solexa now seems to be heading off into Sanger/454 (and soon Pac Bioscience) territory of de novo sequencing with their longer reads. AB has instead pushed for increased numbers of reads, positioning themselves as microarray slayers. Possibly it is the difficulty of pushing ligation-based methods to longer read lengths that has bound AB to their current path.

[1]Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, et al. (2005) Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome. Science 309: 1728-1732.

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Phillip