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Old 01-18-2011, 05:55 PM   #1
seqgirl123
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Default Ligate pcr products for sequencing?

I have read somewhere that PCR products can be turned into libraries using Illumina's genomic sample prep kit and then sequenced on the GAIIx. Can someone explain who would need this kind of sequencing done, and how this all works while making the library? I also read somewhere that the PCR products must be ligated together first in order for library construction and sequencing to work properly, something about the ends of the PCR products having more bias during sequencing, so if they are ligated together it ensures the entire product is read through with less bias. I am not sure if I got that right, I hope someone knows what I am trying to say Also explaining any bias that exist in this kind of library construction will be helpful.

And just to make it clearer, the starting material is PCR products that will be built into libraries, not the usual genomic DNA.

Thanks for clarifications.
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Old 01-21-2011, 02:56 AM   #2
james hadfield
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There is a protocl and a poster on the RainDance site that explains this.

Briefly, a user may amplify 96 loci from 96 individuals and want to seqeucne them all in a single sequencing run. The protocol first lgates PCR products together to for a conatamer. This is fragemetned and goes into the Illumina library prep. The data gets split out at the other end.

Hope this helps.
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Old 01-23-2011, 11:55 AM   #3
Heisman
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This is a strategy for pooled sequencing projects. You have regions of interest and PCR them up in many individuals. Then you pool them together. Then you ligate them all together and sonicate for the reason you stated, to get uniform coverage across all of the strands. If some of the products were 250 base pairs and you made a library prep with them without doing the ligation/sonication, then even with 101 bp paired end sequencing you wouldn't sequence some of the bases. So you can do the ligation/sonication, and then do a normal library prep. You can look at this paper for more information about this: http://genome.cshlp.org/content/earl.../gr.109157.110

This strategy is being replaced by using capture protocols due to the amount of time it takes to do so many PCR reactions, purify the products, and quantify them.
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