Dear all, Many thanks for giving me some encouraging answers, and also for expressing interest in the question whether degraded DNA can be used for genome sequencing of pooled samples. Judging on the few responses it seems possible to get good results from degraded DNA. One might also ask the sequencing provider to prepare a library first, and only proceed with sequencing if the library preparation works out. Thank you also for some good literature suggestions! All the Best, and best of luck with your DNA, Stefan ANSWERS: (1) It really shouldn't be the case that you observe a HMW band for the samples individually, and then not when pooled. To me, this suggests some error when you did the pooling that caused you to lose the sample. When you say "HMW", did you run it out alongside a standard, like lambda DNA? Did they see no band (nothing), or did they see a smear? I'm not sure how you're purifying your DNA, but HMW DNA can sometimes take a long time to dissolve fully. If you run it out and it hasn't dissolved well, it can get stuck up in the wells and might look like a band. When it is later diluted substantially (say, for submission to the facility), it can finally dissolve and may then be smeary if the quality wasn't good. Sometimes residual RNA can cause the smear, too. (2) I recently published a paper where we sequenced >600 paired-end plant samples on HiSeq and GA II platforms. We often found that highly degraded samples yielded a decent number of long scaffolds. My take is that sequencing centers are often too conservative, but it likely also depends on the intended use of the data on whether degradation is a major issue. For more information see my 2012 publication in PLoS ONE available from my website www.evoeco.org. (3) check out the following articles: http://www.biomedcentral.com/1471-2164/14/439 http://www.biomedcentral.com/1471-2164/14/12 Partially degraded samples should work fine. When pooling you should aim pooling samples of simiilar quality in addition to same quantity. (4) 454 rather than illumina, but got some good data out of highly fragmented DNA showing smear on gel from 2kb down. sdennenm@ucalgary.ca