Kevin McKernan, Paul McEwan, Will Morris, Nicole Stange-Thomann, Imani Torruella-Miller, Andrew Sheridan, Alan Wagner, Dudley Wyman, Boris Pavlin, James Benn, Eric S. Lander, Lauren Linton 
Whitehead Institute/MIT Center for Genome Research 
kevinmc@genome.wi.mit.edu 

In an attempt to build a genome center production facility capable of 200Mb finished sequence/year it is important to streamline and simplify operations to allow scalability. At Whitehead Institute we have pursued this goal by designing biochemical protocols that are amenable to automation while constructing the automation as needed. As of recent, we have designed and constructed automation for M13 and plasmid purification. 

Prior to invention of the protocol to be described below, plasmid purification protocols have been labor intensive and expensive. Current commercially available protocols allow 1 tech to process 1000 clones / 8 hour work day at an average cost of $1.20/well (Qiagen). 

M13 purification protocols, on the other hand, have been readily automated in a few laboratories (Stanford, WI) and can produce 12000 clones/ 8 hours per day (15 plates/hour) at a cost of $.07/well while also offering the quality control of full automation. As a result, automating M13 purification has proven to be cost effective since it generates high quality data due to single stranded sequencing. 

Due to these advantages, most genome centers have chosen M13 (Wash U, Whitehead '97, Baylor, Stanford) as their primary vector, supplementing shotgun projects with either 1-2x tiling paths of plasmids to obtain a forward and reverse linked map of the BAC or Cosmid or resorting to directed reverse sequencing of PCR products of their M13 templates. These low coverage double stranded supplements minimize the cost and labor of purifying an entire 10x plasmid subclone library. Unfortunately, PCR based approaches to obtain the forward and reverse linked map with M13 have proven to be problematic in Whitehead's experience due to the difficulties modern polymerases have amplifying repetitive regions and homopolymer stretches. 

A more severe draw back to M13 as a primary vector is its tendency to delete tandem Alu human insert. These deletions still occur using host cells with recA- genotypes(DH5 alpha Laq IQ RecA-) . Isolation of the RF dsDNA of these deleted clones also harbored these deletions suggesting that the deletion events possibly occurs in the rolling circle method of replication where the RF plasmid is temporarily single stranded during replication. 

Plasmids with the pUC Ori seem to be immune to these deletion events and show different cloning bias as assessed by the standard deviation of coverage throughout all plasmid projects. Ideally, where the pass rates, quality, and costs of M13 and plasmids were equivalent, human genome sequencers would choose a double stranded vector system for two primary reasons. 

1) Forward and reverse linkage information provided by the double stranded vectors is invaluable in assembling repetitive genomic DNA. 
2) Double stranded templates can be immediately utilized in a wider variety of biochemical protocols needed for finishing i.e. transposons, shattered libraries, and reverse walks, restriction digests,etc. 

In conclusion, the two vector systems compliment each other. However, supporting both purification pipelines in a high-throughput genome center can be Pyrrhic due the severe differences between the conventional protocols needed to isolate M13 versus plasmid DNAs. Here we describe a novel and automatable plasmid purification protocol that is also compatible for automated M13 DNA purification within the same robotic workstation. 

Thus, enabling a center to freely oscillate between M13 and plasmid DNA template coverage. 

The evolution of the protocol and the status of the robotic workstations will be discussed.