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

2021 ◽  
Vol 22 (20) ◽  
pp. 10908
Author(s):  
Luca Sesta ◽  
Guido Uguzzoni ◽  
Jorge Fernandez-de-Cossio-Diaz ◽  
Andrea Pagnani
Keyword(s):  
Directed Evolution ◽  
High Throughput Screening ◽  
Fitness Landscape ◽  
Selection Process ◽  
Fitness Landscapes ◽  
Wild Type ◽  
Wild Type Sequence ◽  
Mutational Step ◽  
Antibiotic Drug ◽  
Type Sequence

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.

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

2021 ◽  
Author(s):  
Luca Sesta ◽  
Guido Uguzzoni ◽  
Jorge Ferndadez-de-Cossio-Diaz ◽  
Andrea Pagnani
Keyword(s):  
Directed Evolution ◽  
High Throughput Sequencing ◽  
Fitness Landscape ◽  
Fitness Landscapes ◽  
Functional Screening ◽  
Wild Type ◽  
Wild Type Sequence ◽  
Mutational Step ◽  
Antibiotic Drug ◽  
Type Sequence

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.

Wild-Type Sequence ofTP53, Intron 7

2001 ◽  
Vol 155 (4) ◽  
pp. 641-641 ◽  
Author(s):  
Wolfgang Eicheler ◽  
Michael Baumann
Keyword(s):  
Wild Type ◽  
Wild Type Sequence ◽  
Type Sequence

Mutational Analysis of BCR-Abl From Subjects with Relapsed Ph+ALL Treated On the COG Protocol AALL0031: a Report From the Children's Oncology Group.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2634-2634
Author(s):  
Bill H Chang ◽  
Stephanie G Willis ◽  
Linda C. Stork ◽  
Stephen P Hunger ◽  
William L. Carroll ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Lymphoblastic Leukemia ◽  
Intensive Therapy ◽  
Philadelphia Chromosome ◽  
Late Relapse ◽  
Cell Transplant ◽  
Wild Type ◽  
Wild Type Sequence ◽  
Early Results ◽  
Type Sequence

Abstract Abstract 2634 Poster Board II-610 Background: Philadelphia chromosome positive Acute Lymphoblastic Leukemia (Ph+ALL) occurs in 2–5% of pediatric ALL and is associated with a poor prognosis. COG AALL0031 treated children with an intensified chemotherapy backbone plus imatinib. All subjects received imatinib at 340mg/m^2 daily. Exposure to imatinib progressively increased in each of five cohorts. Patients had a total imatinib exposure (before maintenance) of 42 days in cohort 1, 63 days in cohort 2, 84 days in cohort 3, 126 days in cohort 4, and 280 days in cohort 5. All groups received an additional 336 days of imatinib exposure in maintenance cycles 1 through 12 for approximately 2 years (with imatinb given on 21 day cycles for maintenance cycles 1 – 4, and a two-week on/two-week off schedule for maintenance cycles 5 - 12). Early results of this trial show encouraging outcome with a 3-year event free survival of 80±11% (95% CI 64 – 90%) for patients in cohort 5. In studies of adults with Ph+ALL treated with imatinib many patients recurred with imatinib resistant BCR-Abl mutations. To date, there are no data on the occurrence of BCR-Abl mutations in pediatric Ph+ALL. Patients and Methods: We performed nested PCR to identify BCR-Abl point mutations in nine samples obtained at bone marrow (BM) relapse from Ph+ALL subjects on AALL0031. Results: (Table 1) Three samples from cohort 1 that had no exposure to imatinib prior to relapse showed wild-type sequence. There were 5 of 6 samples that also showed wild-type sequence. One sample was from cohort 2 and 3 samples were from cohort 3. Each subject relapsed 1 to 2 years after diagnosis while receiving varying amounts of imatinib with continued intensive therapy. One subject recurred after stem cell transplant in first remission. One sample from cohort 4 recurred after the completion of chemotherapy and imatinib. One subject from cohort 5 carried the histidine 396 to proline (H396P) mutation. This mutation, which increases the imatinib IC50 by 10-fold, has been previously described to occur in adults with CML and Ph+ALL treated with imatinib. The subject from cohort 5 recurred 1 year after diagnosis on therapy with imatinib. Conclusions: Only 1 resistant mutation in BCR-Abl has been identified among nine children with Ph+ALL treated on AALL0031. Therefore, unlike results in the adults, resistant mutations do not appear to drive early recurrence in Ph+ALL. Further studies will be needed to identify whether BCR-Abl mutations are identified in subjects who develop a late relapse after treatment with AALL0031 or subsequent treatment studies. Disclosures: Druker: OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Schultz:novartis: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Improvement in Sensitivity of Allele-specific PCR Facilitates Reliable Noninvasive Prenatal Detection of Cystic Fibrosis

2004 ◽  
Vol 50 (4) ◽  
pp. 694-701 ◽  
Author(s):  
Ourania Nasis ◽  
Shanel Thompson ◽  
Tom Hong ◽  
Margaret Sherwood ◽  
Shawn Radcliffe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Alterations ◽  
Wild Type Allele ◽  
Wild Type ◽  
Type Allele ◽  
Specific Pcr ◽  
Wild Type Sequence ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Type Sequence

Abstract Background: Cell-free fetal DNA circulating in maternal blood has potential as a safer alternative to invasive methods of prenatal testing for paternally inherited genetic alterations, such as cystic fibrosis (CF) mutations. Methods: We used allele-specific PCR to detect mutated CF D1152H DNA in the presence of an excess of the corresponding wild-type sequence. Pfx buffer (Invitrogen) containing replication accessory proteins and Taq polymerase with no proofreading activity was combined with TaqMaster PCR Enhancer (Eppendorf) to suppress nonspecific amplification of the wild-type allele. The procedure was tested on DNA isolated from plasma drawn from 11 pregnant women (gestational age, 11–19.2 weeks), with mutation confirmation by chorionic villus sampling. Results: The method detected 5 copies of the CF D1152H mutant allele in the presence of up to ∼100 000 copies of wild-type allele without interference from the wild-type sequence. The D1152H mutation was correctly identified in one positive sample; the only false-positive result was seen in a mishandled sample. Conclusions: This procedure allows for reliable detection of the paternally inherited D1152H mutation and has potential application for detection of other mutations, which may help reduce the need for invasive testing.

scholarly journals The Ability of Variant Peptides to Reverse the Nonresponsiveness of T Lymphocytes to the Wild-Type Sequence p53264–272 Epitope

2002 ◽  
Vol 168 (3) ◽  
pp. 1338-1347 ◽  
Author(s):  
Thomas K. Hoffmann ◽  
Douglas J. Loftus ◽  
Koji Nakano ◽  
Markus J. Maeurer ◽  
Kazuaki Chikamatsu ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Wild Type ◽  
Wild Type Sequence ◽  
Type Sequence

scholarly journals Adaptation in protein fitness landscapes is facilitated by indirect paths

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Nicholas C Wu ◽  
Lei Dai ◽  
C Anders Olson ◽  
James O Lloyd-Smith ◽  
Ren Sun
Keyword(s):  
Protein Evolution ◽  
Sequence Space ◽  
Fitness Landscape ◽  
Empirical Studies ◽  
Fitness Landscapes ◽  
Complete Subgraph ◽  
Genotype Space ◽  
Subsequent Loss ◽  
Protein Sequence Space ◽  
Type Sequence

The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20L) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 204 = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve.

scholarly journals Mutation Analysis of theMycobacterium leprae folP1Gene and Dapsone Resistance

2010 ◽  
Vol 55 (2) ◽  
pp. 762-766 ◽  
Author(s):  
Noboru Nakata ◽  
Masanori Kai ◽  
Masahiko Makino
Keyword(s):  
Mycobacterium Smegmatis ◽  
Point Mutations ◽  
Wild Type ◽  
Clinical Value ◽  
Nucleotide Substitutions ◽  
Recombinant Bacteria ◽  
Wild Type Sequence ◽  
The Relationship ◽  
Line Drug ◽  
Type Sequence

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.

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