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Old 09-14-2017, 04:36 AM   #4
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Location: Brisbane

Join Date: Oct 2009
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Originally Posted by tomlodz View Post
Hi everyone,

We try to do NGS on ancientDNA according to the protocol of this publication:

Can you please provide some advice? What cause such difference in quality of reads for this two RUNS. Most of the parameters are included in the attached pdf file. Thanks a lot!
I think a lot of the run difference can be explained by as you state: issues with quantification. You've included the MiSeq run stats, but I think there's a more important question - what did the library QC looks like (BioAnalyser/Gel)?Ancient DNA is massively variable in terms of quality and quantity. Were you able to visualise the DNA in extracts to determine mean size length prior to library prep? Often there is high molecular weight DNA in aDNA samples as well. A large variation in size of the library will impact qPCR and clustering. You should be able to easily see an ancient DNA library on a BioAnalyser chip post-PCR.

Endogenous DNA in truly ancient samples is always very low unless you enrich for it. The first Neanderthal genome sequence runs were around 90% microbial.

As a general rule - read 2 (second seq read) also is nearly always rubbish in ancient DNA for me on a HiSeq. I maybe get 20-30 bases before it turns to rubbish. I do know of a few possible reasons for this but won't go into detail to save text.

What is the concentration of adapters are you using? If there's not much DNA then the advised adapter concentration should be dropped to prevent dimers and artefact. Are you sure your aDNA is still double stranded? Meyer et al found a lot of single stranded DNA in their really old material and actually designed a new protocol to be able to sequence this. If your aDNA is really damaged it won't work very with the t4 ligation.

This protocol you are using is 7 years old and was designed for modern material (and derived from Meyer's et al. work on the 454). Are you changing anything e.g. this protocol shears the samples which is not advisable for most ancient DNA> A lot of this is no longer standard, even for modern material. Meyer and others have updated protocols to work much better for ancient DNA. You can also create ancient DNA libraries that don't require custom sequencing primer thus immediately removing a huge component of variability. And on that note - was your sequencing primer HPLC purified?

A 2x250bp run will result in most ancient DNA sequencing reads being full of adapter. Again - look at the library distribution in QC. Adapter sequence will mess up quality due to being low diversity etc. You're also paying for sequencing you're just going to throw out. On a Hiseq people will wear this when it's only a lane or two, but a MiSeq enables you to choose read lengths and customise a lot better and cut down work on the bioinformatics end.
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