Allpaths-Lg, Using Sanger Data To Create The Fragment Library

Question

[David M]

We're currently looking at different assemblers, and I'm in the process of learning about/testing ALLPATHS-LG. At the present we have 14 lanes of illumina data (for a mammalian-sized genome), but don't yet have the fragment (= superread) library. Could I still get a decent assembly by artificially creating the fragment library from a library of Sanger reads?

Thanks!

[David M]

Anybody have any ideas if this is a problem?

Answer 1

[Torst]

This would probably work, as long as you have (1) high quality across the whole read (2) enough coverage. For (1) the super-read is constructed by overlapping alignment of 100bp reads from a 180bp fragment, so that the lower quality 3' ends of each read overlap to produce an overall higher quality 180bp read. For (2) I think you could be in trouble, as you won't get the 50x required from a Sanger library, unless you shred your Sanger reads into overlapping 180bp reads to the sufficient depth. In this case it would probably be fine.

[David M]

If I shred the Sanger reads into overlapping reads, wouldn't I be lending a statistical certainty to the new reads that doesn't exist? Ie, If a sanger base is incorrect, and that base is used in 50 'fake' reads, don't I grant certainty to an incorrect base where it shouldn't exist?

[Torst]

Does ALLPATHS use quality scores at all? If not, if there is a base with low quality in your Sanger read, you could replace it with "N" to mark it for correction/PCR-check later. If so, perhaps scale all your Q scores in the Sanger reads by the factor appropriate for the shredding depth, and ALLPATHS will treat them more correctly.