Archive for September, 2010

A Favorite Rothian Story

Tuesday, September 28th, 2010

An extravagantly wealthy king had twin sons. One was very pessimistic and the other unusually optimistic. The king was worried that both would suffer in the long run from these defects. He decided to apply the well-known therapy of confronting extremes.

He led his young pessimist to a large room in the palace, filled with wonderful toys from floor to ceiling. The child immediately burst into tears. “What is the matter, my son?”, exclaimed the king. “I know that these toys will all eventually break, and my sadness will be all the greater as there are so many!”, exclaimed the little prince through his tears.

Puzzled by this, the king fetched his other son, the eternal optimist, and led him to a large room in the royal stables, a room filled from floor to ceiling with manure. When the child saw this, he immediately yelled a cry of glee, ran to the pile and began to dig furiously through it with his bare hands. “My son!”, said the king, “Have you gone mad? Why are you doing this?”, to which the joyous prince replied, “Oh, father! Thank you so much! With all this horse shit, there’s sure to be a pony in there somewhere!”

-- Eric Kofoid

The Sad Truth about Illumina Data Clustering

Tuesday, September 28th, 2010

This article is a continuation of Illumina Data Clustering, and is a perfect example of why we, as scientists, should resist the hubris of premature expectations.

The standard Illumina protocol for library preparation requires 18 cycles of PCR after adaptor ligation to enrich for fragments with doubly modified ends. Incomplete products from a previous round can snap-back during this step (“megaprimer snap-back”), creating artifactual templates which will then amplify along with the others. This is a first order process and should be fairly common when the 3′ of the elongating strand just happens to fall on a complementary REP site. A less common artifact could occur by a second order process involving megaprimer extension and reannealling in trans to a complementary REP site.

I found a group of closely spaced NlaIV restriction sites which would destroy megraprimer formation by the snap-back route when digested. If clusters arose from preexisting TIDs or by the rare megaprimer extension event, NlaIV digestion would have no effect on cluster amplification.

I did the experiment, and found that cutting the template DNA with NlaIV prevented amplification. I am forced to conclude that the beautiful clustering of REP-mediated TID joints found in our data is strictly man made by megaprimer snap-back!

Models for PCR Detection of REP-Mediated Clusters

-- Eric Kofoid