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Old 03-13-2014, 11:30 AM   #1
jlayer
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Location: Boston

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Smile Sequencing Translocation Junctions

Hello All,

I'm a 2nd year graduate student studying DNA repair. I want to sequence an amplicon library of translocation junctions to quantify the amount of deletions, insertions, and microhomology at breakpoints.

My question is after obtaining the sequencing reads, is it customary to deduplicate reads in the sequencing files? This seems counterintuitive to me since an amplicon based library should contain many identical reads and leave me unable to quantitate different types of junctions.

Any suggestions for packages to use for cleaning these types of reads and/or statistical analysis would be appreciated!

Thanks!
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Old 03-23-2014, 08:08 AM   #2
ECO
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Are you sequencing inverse PCR products? How are you generating the amplicons?

I ask because if you want the best quantitation option to help you deal with dups, you should explore a way to add a molecular barcode to each translocation event during the library generation process (prior to PCR). Check out this paper and the previous one it references: http://www.ncbi.nlm.nih.gov/pubmed/24449890

Basically you ligate a high complexity tag prior to PCR to each molecule, thus you can distinguish PCR duplicates from duplicate events/observations.
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