qPCR curves

Each line on the graph below represents a qPCR for one of our sub-pools (each sequence we are targeting represents a sub-pool: CB top, CB bottom, FH top, FH bottom, Myc 198, Myc 981). By plotting the relative flouresence present in each cycle, we can visualize the pogress of the reaction. When performing a qPCR, one wants to run the reaction while the growth is still exponential. During this phase, all the oligos are replicated at equal rates. Once growth begins leveling off, as it does at the end of the graph below, the reaction should be stopped as the oligos are now being replicated unequally.

Based on the graph and gel below, we knew we successfully amplified each of our target sub-pools.

Because flourescence grows exponentially and just begins to level off at the end of the graph (where PCR was terminated), we are confident that overamplification did not occur, so individual oligos in each of our sub-pools were amplified at roughly equal rates. This preserves library integrity.

After a PCR clean up, we ran a gel in order to confirm that the qPCR produced the expected product, of approximately 130 bps. Based on the gel, we could conclude that our qPCR was indeed successful, and our oligo library was ready to use.

Sequencing results of Library transformation

Error rates

• Perfect sequence matches a designed ZF: 57.1% (44/77)
• Single SNP: 2.6% (2/77)
• Two or more SNPs: 18.2% (14/77)
• Frame shift: 22.1% (17/77)

We determined the overall per base pair error rate for this set sequenced to be around 1/200, which includes errors generated by the chip, or generated during PCR and assembly. These are a bit higher than those found by Kosuri, et al., but within a reasonable margin.

Distributions

Of the 77 samples with good sequencing results, 2 sequences were repeated once. Discounting these, 73 of the 77 sequences, or 94.8%, were unique, showing substantial variability within the library.