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.