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Old 05-29-2014, 07:27 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by dtm2451 View Post
Hello,

I am working on a deep sequencing protocol for a PCR amplicon (~650bp) using the TruSeq DNA PCR-Free Sample Preparation Kit and I am seeing extra peaks in my final bioanalyzer traces that concern me because I don't know what they might come from.

Peaks on the trace:
+Small peak at size of original insert
+Medium-sized peak that I think might correspond to insert+1adapter
+Large peak that I think might correspond to insert+2 adapters
-mini peak to the right of the "insert" peak
-mini peak to the right of the "insert+1adapter" peak
-mini peak to the right of the "insert+2adapters" peak

Does anyone know what the last three peaks might be?

I am attaching both the TapeStation on the initial PCR material and the bioanalyzer on the final library.

Thanks!
Dan
Hi Dan,

Illumina adapters are about each 60 bases long.

But you may be right. Illumina kit adapters are "Y"-adapters with only about 10 bp of doublestranded DNA and the rest (~50 bases) as double single-stranded tails. Single stranded molecules tend to migrate slower on Agilent chips than corresponding length double stranded molecules. So those Y-adapters may be introducing some drag.

But that would be a lot of drag from a few hundred bases of double ssDNA. Seems unlikely to me.

Another hypothesis would be that the "1539 bp" fragment is migrating slowly because the ligase is still attached to the amplicon.

Another possibility (this is the one I like) is that the "901 bp" fragment has both adapters ligated and is running only a little larger than its expected because of the Y-adapters: 656+120=776bp double-stranded length . The 1539 bp fragment would have a double insert, 656+656+120=1432 which would fit pretty well. That would suggest the A-tailing step did not work well -- left a substantial percentage of the ends blunt.

You posted this question long ago, so you could probably update us on your results. But if you used this library, it probably worked okay. For reasons unclear to me, short amplicons seem to cluster vastly better than longer amplicons. So your data set would remain fairly free from chimerics.

--
Phillip
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