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Old 02-02-2012, 12:23 AM   #1
xanqueta
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Hello all,
I am working on acientDNA, I have experience on isolating genetic material from archaeological samples....sometimes a ungrateful work!
My lab wants prepare some NGS libraries to run them on Illumina GA/HiSeq and I'm searching a protocol to prepare a library from my very fragmented sample (50-200 bp genomic DNA). I don't see many posts on aDNA and I would like to know if anybody has experience on this or with your knowledge on DNA sample prep you have tips that could help me.
Any suggestions are very welcome.
thanks!
Cheers
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Old 02-05-2012, 05:52 AM   #2
yaximik
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I would start from analysing Paabo's papers. I work with ancient DNA using 454 and it is nightmare. The most difficult problem is not libary prepration, it is quantification of libraries. Perhaps qPCR is the only way, but uncontrolled fragment distribution in fragmented DNA makes it much less reliable as compared to libraries made from gDNA fragmented in controlled way. And the contamination problem represents much worse challenge as compared to analysis of aDNA by PCR.
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Old 02-14-2012, 12:43 AM   #3
xanqueta
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Dear Yakimik,
thanks a lot for your reply!! I'ts always nice to hear someone that has same aDNA nightmares...
Anyway, I'm also worried about the sampleprep itself. With Illumina I have 3 purification steps after end repair, a-tail and ligation. This means I'll lost lot of starting material. If I'm not wrong with 454 you have one step less. I'm also worried about the PCR step. Phusion Hifi enzyme (used in Illumina library enrichment) is not very robust enzyme. I used to use golden taq for my normal aDNA PCR….
I’ll follow your advice; I’ll use qPCR to quantify my library.
Cheers!!!
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Old 02-14-2012, 04:29 AM   #4
yaximik
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You're welcome. I do not use Phusion, it is a crappy enzyme in my opinion. Never could make it work. KOD Hot Start from Novagen is much better, essentially the same protocol can be used. Recently, KAPA Biosystems offered a similar enzyme, it is even more active, needs some adjustments. KAPA also offers platform-specific qPCR kits for library quantification, my experience was good, but it is too expensive in my opinion. Similar performance I got from more reasonably priced Takara's SYBR exTaq mix (also from Novagen) with standard emPCR primers (454 platform), but I guess Illumina's standard primers can be used too.
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Old 02-22-2012, 12:31 PM   #5
odile
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Hi xanqueta,

I also work with aDNA. The library prep for 454 is indeed quicker than the Illumina’s but the sequencing part is very long (tons of washes in particular). I prefer Illumina by far. The size of the reads also matters. If your DNA is severely degraded, then go with Illumina. If it's above 200bp, you can try 454. I personally like the high throughput of Illumina. Each library give ~ 30 millions reads, ie great coverage. Of course, there is a lot of bacterial/fungal DNA in there, but that's another problem. As suggested before, I'd review literature. Leipzig started with a few great protocols but there are many more labs that publish NGS on ancient DNA nowadays. Good luck. Ancient DNA can truly be frustrating but when it works, nothing feels better
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Old 02-23-2012, 03:40 AM   #6
yaximik
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Odile,

I wonder what do you use to filter out bacterial/fungal sequences? I could not find something generic, way to much diversity, but going essentially manually over 30 million reads with BLAST is unimaginable either.
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Old 02-23-2012, 05:29 AM   #7
odile
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I do some target enrichment using human mtDNA specific probes. This way I get the entire human mtgenome with a very good coverage.
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