AMaLa: Analysis of Directed Evolution Experiments via Annealed Mutational Approximated Landscape

We present Annealed Mutational approximated Landscape (AMaLa), a new method to infer fitness landscapes from Directed Evolution experiments sequencing data. Such experiments typically start from a single wild-type sequence, which undergoes Darwinian in vitro evolution via multiple rounds of mutation and selection for a target phenotype. In the last years, Directed Evolution is emerging as a powerful instrument to probe fitness landscapes under controlled experimental conditions and as a relevant testing ground to develop accurate statistical models and inference algorithms (thanks to high-throughput screening and sequencing). Fitness landscape modeling either uses the enrichment of variants abundances as input, thus requiring the observation of the same variants at different rounds or assuming the last sequenced round as being sampled from an equilibrium distribution. AMaLa aims at effectively leveraging the information encoded in the whole time evolution. To do so, while assuming statistical sampling independence between sequenced rounds, the possible trajectories in sequence space are gauged with a time-dependent statistical weight consisting of two contributions: (i) an energy term accounting for the selection process and (ii) a generalized Jukes–Cantor model for the purely mutational step. This simple scheme enables accurately describing the Directed Evolution dynamics and inferring a fitness landscape that correctly reproduces the measures of the phenotype under selection (e.g., antibiotic drug resistance), notably outperforming widely used inference strategies. In addition, we assess the reliability of AMaLa by showing how the inferred statistical model could be used to predict relevant structural properties of the wild-type sequence.

AMaLa: analysis of Directed Evolution experiments via Annealed Mutational approximated Landscape

We present Annealed Mutational approximated landscape (AMaLa), a new method to infer fitness landscapes from Directed Evolution experiment sequencing data. Directed Evolution experiments typically start from a single wild-type sequence, which undergoes Darwinian in vitro evolution acted via multiple rounds of mutation and selection with respect to a target phenotype. In the last years, Directed Evolution is emerging as a powerful instrument to probe fitness landscapes under controlled experimental condition and, thanks to the use of high-throughput sequencing of the different rounds, as a relevant testing ground to develop accurate statistical models and inference algorithms. Fitness landscape modeling strategies, either use as input data the enrichment of variants abundances and hence require observing the same variants at different rounds, or they simply assume that the variants at the last sequenced round are the results of a sampling process at equilibrium. AMaLa aims at leveraging effectively the information encoded in the time evolution of all sequenced rounds. To do so, on the one hand we assume statistical sampling independence between sequenced rounds, and on the other we gauge all possible trajectories in sequence space with a time-dependent statistical weight consisting of two contributions: (i) a statistical energy term accounting for the selection process, (ii) a simple generalized Jukes-Cantor model to describe the purely mutational step. This simple scheme allows us to accurately describe the Directed Evolution dynamics in a concrete experimental setup and to infer a fitness landscape that reproduces correctly the measures of the phenotype under selection (e.g. antibiotic drug resistance), notably outperforming widely used inference strategies. We assess the reliability of AMaLa by showing how the inferred statistical model could be used to predict relevant structural properties of the wild-type sequence, and to reproduce the mutational effects of large scale functional screening not used to train the model.

A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum

Introduction. Macrolides could be a potential alternative treatment for Treponema pallidum infections in patients; however, macrolide-resistant T. pallidum is spreading rapidly worldwide. Hypothesis/Gap Statement. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that T. pallidum cannot be cultured in vitro. Aim. In this study, we constructed a method for rapidly identifying T. pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs). Methodology. A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild-type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3′ end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction. Results. The assay had an equivalent detection limit of 1.0×101 copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 µM PNA probe, LAMP amplified up to 1.0×101 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1.0×103 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays. Conclusion. We developed a PNA-mediated LAMP method that enabled us to rapidly identify T. pallidum and determine its macrolide susceptibility via a culture-independent protocol.

An ultra-sensitive multiplexed enrichment of rare DNA variants via toehold exchange principle.

e13658 Background: As a major type of liquid biopsy, circulating tumor DNA (ctDNA) provides important somatic mutation information, but the feature of rare allele frequency of mutation in ctDNA hampers its wide application. Methodology with high sensitivity, cost effectiveness, and multiplexing capability is needed for analyzing ctDNA samples. Methods: We have developed a novel methodology, termed MTEA (Multiplexed Toehold Exchange Amplification), which selectively amplifies and enriches all sequence variants (including SNV and deletion) 1000-fold over the wild-type sequence within ~20-nucleotide window. The amplified products are detected by Sanger sequencing or next-generation sequencing (NGS). This novel approach takes advantage of toehold exchange principle in which PCR amplification of wild-type rather than mutants would be greatly reduced due to the formation of a duplex of wild-type sequence with a blocker targeting to the wild-type rather than the mutant alleles. Results: Using circulating cell-free DNA (cfDNA) reference standards, we demonstrated that the major driver mutations of lung adenocarcinomas, including EGFR L858R and exon 19 deletion, KRAS G12X, and BRAF V600E, were enriched and detected simultaneously with MTEA technology at abundance as low as 0.01-0.05%. The clinical implications of MTEA technology were further validated using ctDNA from liquid biopsy specimens of 29 non-small cell lung cancer patients, and results were compared with those collected from the NGS and ddPCR analysis. Consistent results were obtained from all of the three platforms, which indicated 100% accuracy. The MTEA method has advantages over ddPCR since the later lacks the multiplexing detection ability. The MTEA technology can also save cost compared with NGS method. Conclusions: MTEA technology can enrich ctDNA from cancer patients to detect ultra-low abundance of clinically relevant mutations. Upon validation using a larger cohort, the technology may have wide application in precision medicine and guide clinicians for therapeutic decisions.

SINGLe: Accurate detection of single nucleotide polymorphisms using nanopore sequencing in gene libraries

Nanopore sequencing is a powerful single molecule DNA sequencing technology which offers high throughput and long sequence reads. Nevertheless, its high native error rate limits the direct detection of point mutations in individual reads of amplicon libraries, as these mutations are difficult to distinguish from the sequencing noise.In this work, we developed SINGLe (SNPs In Nanopore reads of Gene Libraries), a computational method to reduce the noise in nanopore reads of amplicons containing point variations. Our approach uses the fact that all reads are very similar to a wild type sequence, for which we experimentally characterize the position-specific systematic sequencing error pattern. We then use this information to reweight the confidence given to nucleotides that do not match the wild type in individual variant reads. We tested this method in a set of variants of KlenTaq, where the true mutation rate was well below the sequencing noise. SINGLe improves between 4 and 9 fold the signal to noise ratio, in comparison to the data returned by the basecaller guppy. Downstream, this approach improves variants clustering and consensus calling.SINGLe is simple to implement and requires only a few thousands reads of the wild type sequence of interest, which can be easily obtained by multiplexing in a single minION run. It does not require any modification in the experimental protocol, it does not imply a large loss of sequencing throughput, and it can be incorporated downstream of standard basecalling.

The hTERT core promoter forms three parallel G-quadruplexes

The structure of the 68 nt sequence with G-quadruplex forming potential within the hTERT promoter is disputed. One model features a structure with three stacked parallel G-quadruplex units, while another features an unusual duplex hairpin structure adjoined to two stacked parallel and antiparallel quadruplexes. We report here the results of an integrated structural biology study designed to distinguish between these possibilities. As part of our study, we designed a sequence with an optimized hairpin structure and show that its biophysical and biochemical properties are inconsistent with the structure formed by the hTERT wild-type sequence. By using circular dichroism, thermal denaturation, nuclear magnetic resonance spectroscopy, analytical ultracentrifugation, small-angle X-ray scattering, molecular dynamics simulations and a DNase I cleavage assay we found that the wild type hTERT core promoter folds into a stacked, three-parallel G-quadruplex structure. The hairpin structure is inconsistent with all of our experimental data obtained with the wild-type sequence. All-atom models for both structures were constructed using molecular dynamics simulations. These models accurately predicted the experimental hydrodynamic properties measured for each structure. We found with certainty that the wild-type hTERT promoter sequence does not form a hairpin structure in solution, but rather folds into a compact stacked three-G-quadruplex conformation.

Mutation Analysis of theMycobacterium leprae folP1Gene and Dapsone Resistance

ABSTRACTDiaminodiphenylsulfone (dapsone) has long been used as a first-line drug worldwide for the treatment of leprosy. Diagnosis for dapsone resistance ofMycobacterium lepraeby DNA tests would be of great clinical value, but the relationship between the nucleotide substitutions and susceptibility to dapsone must be clarified before use. In this study, we constructed recombinant strains of cultivableMycobacterium smegmatiscarrying theM. leprae folP1gene with or without a point mutation, disrupting their ownfolPgene on the chromosome. Dapsone susceptibilities of the recombinant bacteria were measured to examine influence of the mutations. Dapsone MICs for most of the strains with mutations at codon 53 or 55 ofM. leprae folP1were 2 to 16 times as high as the MIC for the strain with the wild-typefolP1sequence, but mutations that changed Thr to Ser at codon 53 showed somewhat lower MIC values than the wild-type sequence. Strains with mutations at codon 48 or 54 showed levels of susceptibility to dapsone comparable to the susceptibility of the strain with the wild-type sequence. This study confirmed that point mutations at codon 53 or 55 of theM. leprae folP1gene result in dapsone resistance.