4. Discussion
LASSO technology enables massively multiplexed capture of large DNA
fragments for sequencing and/or expression cloning. A key factor for
LASSO capture efficiency is the quality of the LASSO probe library in
terms of probe sequence identity and probe representation. A previous
assembly protocol we developed presented a number of drawbacks that
resulted in a poor quality of the LASSO library with only
~10% of correctly assembled LASSO probes. The critical
phase was the self-circularization by ligation of the LASSO probe
precursor, wherein EcoRI digested probe ends were intramolecularly
ligated to each other. Shukor S et al. [34] noted
that when attempting to generate thousands of probes in a single
reaction by this manner, there exists a strong possibility that
intermolecular ligations would manifest as mismatched probe arms on a
mature LASSO probe. The presence of the discordant probes in the mature
LASSO libraries was responsible for a reduction of the efficiency,
likely due to the depletion of PCR reactants used up for the
amplification of low molecular weight unspecific DNA amplicons arising
in the post capture PCR (Supplementary figure 1 table a).
To improve the quality of the LASSO probe libraries we developed a
different LASSO assembly methodology that leads to the same mature LASSO
probe configuration but uses the cre recombination of a custom plasmid
(pLASSO) to supply the linker of the mature LASSO probe. The assembly
process herein described starts with the cloning of the pre-LASSO
library in the pLASSO vector and E.coli transformation. The
multiplication of a pLASSO library in E.coli, reduces the possible
skewing of the different LASSO probes in the library. The plasmid
library was then subjected to Cre recombination of the two loxp sites
oriented head-to tail in pLASSO resulting in the excision of a DNA
minicircle containing the LASSO precursor in its final configuration
(Figure 2).
The LASSO library we assembled targeted the same E. coli ORFs of our
previous work [33] and the LASSO probes had
identical design but displayed superior capture performance. This
observation is in agreement with the higher percentage
(~46%) of concordant probes present in the E.coli LASSO
library. The median RPKM for targeted ORFs versus untargeted ORFs was
much higher than produced with the previous LASSO assembly methodology -
especially for shorter ORFs (~8 times higher). This
finding suggests that a better quality of the LASSO probe library
results in a higher capture efficiency and in the reduction of
undesirable low molecular weight amplicons in post capture PCR
(Supplementary Figure 1)
With a model system, we showed that the sensitivity of LASSO capture
potentially allows for the massive parallel capture of DNA targets at
the whole human genome scale. We also evaluated the ability of the new
LASSO probes to capture two individual full-length ORFs from a total
human cDNA. The genes β-actin and glyceraldehyde 3-phosphate
dehydrogenase were captured thereby verifying that this new LASSO
production method provides comparable capture efficacy to the
first-generation method using a self-circularization reaction. Future
evaluation of this method is necessary to evaluate the breadth of this
LASSO technology for the creation of human protein libraries.
As novel long-read sequencing technologies emerge, there is an
increasing need for novel target enrichment methods that allow highly
multiplexed selection of kilobase-sized DNA. We expect that LASSO probes
can find immediate applications for targeted construction of long-read
sequencing libraries. LASSO probes can also be used for the rapid and
inexpensive production of pooled ORFeome libraries that can be expressed
using standard vectors for functional screening applications.