Friday, April 4, 2008

Lab work without data analysis and management is doo doo

As we begin to contemplate next generation sequence data management, we can use Sanger sequencing to teach us important lessons. One of which, is the value of linking laboratory and data workflows to be able to view information in the context of our assays and experiments.

I have been fortunate to hear J. Michael Bishop speak on a couple of occasions. He ended these talks by quoting one of his biochemistry mentors, "genetics without biochemistry is doo doo." In a similar vein, lab work without data analysis and management is doo doo. That is when you separate the lab from the data analysis, you have to work through a lot of doo to figure things out. Without a systematic way to view summaries of large data sets, the doo is overwhelming.

To illustrate, I am going to share some details about a resequencing project we collaborated on. We came to this project late, so much of the data had been collected, and there were problems, lots of doo. Using Finch however, we could quickly organize and analyze the data, and present information in summaries with drill downs to the details to help troubleshoot and explain observations that were seen in the lab.

10,686 sequence reads: forward / reverse sequences from 39 amplicons from 137 individuals

The question being asking in this project was: are there new variants in a gene that are related to phenotypes observed in a specialized population? This is the kind of question medical researchers ask frequently. Typically they have a unique collection of samples that come from a well understood population of individuals. Resequencing is used to interrogate the samples for rare variants, or genotypes.

In this process, we purify DNA from sample material (blood), and use PCR with exon specific probes to amplify small regions of DNA within the gene. The PCR primers have regions called universal adaptors. Our sequencing primers will bind to those regions. Each PCR product, called an amplicon, is sequenced twice, once from each strand to give double coverage of the bases.

When we do the math, we will have to track the DNA for 137 samples and 5343 amplicons. Each amplicon is sequenced, at a minimum twice, to give us 10,686 reads. From a physical materials point of view that means 137 tubes with sample; 56, 96-well plates for PCR; and 112, 96-well plates for sequencing. In a 384-well format we could have used 14 plates for PCR and 28 plates for sequencing. For a genome center, this level of work is trivial, but for a small lab this is significant work and things can happen. Indeed as not all the work is done in a single lab the process can be more complex. And you need to think about how you would lay this out - 96 does not divide by 39 very well.

From a data perspective, we can use sequence quality values to identify potential laboratory and biological issues. The figure below summarizes 4608 reads. Each pair of rows is one sample (forward / reverse sequence pairs, alternating gray and white - 48 total). Each column is an amplicon. Each cell in the table represents a single read from an amplicon and sample. Color is used to indicate quality. In this analysis, quality is defined as the ratio of Q20 to read length (Q20/rL), which works very well for PCR amplicons. The better the data, the closer this ratio is to one. In the table below, green indicates Q20/rL values between 0.60 and 1.00, blue indicates values between 0.30 and 0.59, and red indicates Q20/rL values less than 0.29. The summary shows patterns that, as we will learn next week, show lab failures and biological issues. See if you can figure them out.

2 comments:

Eric said...

Hi Todd,

Great post!

I'll take a couple of stabs at the patterns.

First the easy one...samples 1 and 4 clearly were either not dispensed properly or contain a ton of inhibitors that ruined all their PCRs.

Second, amplicons 24 and 27 could either have bad F & R sequencing primer design, respectively, or...the F & R reads for them got swapped, and one of the two amplicons just sequences poorly.

That's all I've got...looking forward to reading the next installment! :D

Aarthy said...

Great post Todd. Amplicons 7, 24 and 27 seem to either sequence poorly in one or both directions. This may mean primer design issues, high GC content and or repetitive sequences in the amplicon. Eagerly awaiting the next post!!

Aarthy