Figure 2 , Schematics of DNA recombinase mediated LASSO
probe library assembly . a , A ssDNA pre-LASSO oligonucleotide
library is converted to double stranded DNA form by PCR using primer
selectors. b , The selected pre-LASSO library in shuttled in the
linearized pLASSO vector and used for transformation in E.coli .c , Gel electrophoresis of BamHI and SalI digested library, lane
1 illustrate presence of a DNA band correspondent with the pre-LASSO
insert indicating successful cloning of the pre-LASSO library. lane 2,
pLASSO alone. d , The native supercoiled pLASSO library is
digested nicking with endonuclease and subjected to Cre recombination
that generates a DNA minicircle containing the pre-LASSO and a circular
2.7 kb DNA circle. The 2.7kbDNA circle, together with the unreacted
plasmids and bigger DNA circles generated by inter-plasmid recombination
(not shown in the drawing) are eliminated by restriction followed by
exonuclease digestion. f , Gel electrophoresis illustrates in
lane 1 successful formation of DNA minicircles (orange arrow) together
with the 2.7 kb circular DNA remaining parts of pLASSO (green arrow),
the unreacted plasmid (blue arrow). The approximately 6kb band (yellow
arrow) correspond to the recombination of two different plasmids
(inter-plasmid recombination). In lane 2, When using an un-nicked pLASSO
library for Cre recombination the DNA band correspondent to DNA
minicircle was absent. e , inverse PCR. g , maturation
of the LASSO probe library by removal of primer annealing sites and
digestion of a DNA strand. h, Gel electrophoresis, in lane 1
inverted PCR amplicon correspondent to the linearized minicircle. In
lane 2 negative control for Cre-recombination. Legend. Sal1,
BamH1 and BspQ1 indicate restriction enzyme sites, nick indicates
nicking endonuclease site NtBspQ1 the * indicates phosphorothioate
bonds, U indicate a deoxyuracil moiety. L1 indicates 1 kb DNA Ladder
(NEB), L2 indicates Low Molecular Weight DNA Ladder (NEB)
An approximately 6kb DNA band (yellow arrow) was also seen in the final
product and likely corresponded to the size of pLASSO concatemers. The
formation of circular concatemers was likely caused by to the
recombination of loxP sites located in different pLASSO molecules
(inter-molecular recombination). In order to eliminate the recombination
products other than the DNA minicircles, we performed a digestion with
SwaI restriction enzyme (SwaI recognition sequence present in the 2.7 kb
circular DNA, pLASSO substrate and pLASSO concatemers) followed by
Exonuclease V digestion (Fig. 2d ). We also performed the
Cre-recombination reaction using the un-nicked pLASSO library; in this
case the DNA band correspondent to DNA minicircle (red arrow inFig. 2 f lane 1) was not visible (Fig. 2 f, lane 2).
This observation suggests that the uncoiled form of pLASSO plasmid,
induced by the DNA nick, was a better substrate for Cre-recombinase that
the natural supercoiled un-nicked form. The minicircles were
subsequently subjected to inverse PCR (Fig. 2 e ) using primers
that anneal on the inverse PCR primer-annealing site. The expected size
of the inverted PCR product was verified in agarose gel (Fig. 2
h ). The negative control for Cre recombination showed no inverted PCR
amplicon was present (gel lane 2) indicating that DNA minicircles were
not formed in absence of Cre recombinase and that pLASSO was completely
digested.
The inverted PCR product is in the final mature LASSO probe
configuration with the annealing arms flanking the conserved region
(blue) derived from pLASSO (Fig. 2 g ). The external primer
sites were then removed by digesting with BspQ1 restriction enzyme
followed by exonuclease digestion and treatment with USER enzyme as
described in Material and Methods. The final mature LASSO probe library
was then ready for massively parallel target capture reactions.
To assess quality and uniformity of the LASSO library produced we
performed NextSeq 150bp paired end sequencing of the E.coli LASSO
library at the inverted PCR stage (Fig 2 g ) in which ligation
and extension arms are already coupled with the conserved DNA backbone
in the final configuration.
Analysis of NGS data reveal that the majority of LASSO probes were
composed of ~45% correctly paired probe arms versus
total read sequences per probe type (Fig. 3 a) . The mean for
correctly paired probes as a ratio of concordant probes vs discordant
probes calculated for all LASSO probes was 46 %.
Syukri S. and coworkers (2019) when assessing the
quality of the E.coli LASSO library reported only 10% of concordant
probes when using our previous assembly methodology (Tosi et al.,2017). As shown in Fig. 3b , the majority of the probes were
present within two tenfold the normalized abundance of the median
indicating a relatively uniform representation of LASSO probes in the
LASSO library.