EasyDIVER: A Pipeline for Assembling and Counting High-Throughput Sequencing Data from In Vitro Evolution of Nucleic Acids or Peptides

Blanco, Celia; Verbanic, Samuel; Seelig, Burckhard; Chen, Irene

doi:10.1007/s00239-020-09954-0

Cited by 15 publications

(16 citation statements)

References 13 publications

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“…The raw, paired-end, demultiplexed Illumina read files (i.e., FASTQ files) of all k -Seq samples were processed with the EasyDIVER pipeline to create count files containing dereplicated lists of the central variable region sequences and their count reads ( 106 ). Every unique sequence detected in the count file of the input sample was tracked across all k -Seq samples.…”

Section: Methodsmentioning

confidence: 99%

“…First, we examined whether any novel ribozyme families arose in either selection condition. The HTS result of each selection pool was analyzed by EasyDIVER pipeline ( 106 ) to create dereplicated lists of the central variable region sequences and their count reads. The sequences on the list were clustered into a peak if the sequences were within a Hamming distance of ≤3 from the known family wild types (S1a, S1b, S2a, S2b, and S3).…”

Section: Methodsmentioning

confidence: 99%

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Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

Lai

Liu

Chen

2021

Proc. Natl. Acad. Sci. U.S.A.

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Functional biomolecules, such as RNA, encapsulated inside a protocellular membrane are believed to have comprised a very early, critical stage in the evolution of life, since membrane vesicles allow selective permeability and create a unit of selection enabling cooperative phenotypes. The biophysical environment inside a protocell would differ fundamentally from bulk solution due to the microscopic confinement. However, the effect of the encapsulated environment on ribozyme evolution has not been previously studied experimentally. Here, we examine the effect of encapsulation inside model protocells on the self-aminoacylation activity of tens of thousands of RNA sequences using a high-throughput sequencing assay. We find that encapsulation of these ribozymes generally increases their activity, giving encapsulated sequences an advantage over nonencapsulated sequences in an amphiphile-rich environment. In addition, highly active ribozymes benefit disproportionately more from encapsulation. The asymmetry in fitness gain broadens the distribution of fitness in the system. Consistent with Fisher’s fundamental theorem of natural selection, encapsulation therefore leads to faster adaptation when the RNAs are encapsulated inside a protocell during in vitro selection. Thus, protocells would not only provide a compartmentalization function but also promote activity and evolutionary adaptation during the origin of life.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

Lai

Liu

Chen

2021

Proc. Natl. Acad. Sci. U.S.A.

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“…After purification with the QiaQuick PCR Purification Kit (Qiagen), the enriched DNA pool was transcribed (see Supplementary Data) and subjected to an additional round of selection (black arrow Figure 1B ). After six rounds of selection, the G6 DNA pool was analyzed by high-throughput Illumina sequencing (Illumina MiSeq, nano output) and the resulting sequence data was processed using EasyDIVER ( 41 ) ( Supplementary Materials and Methods and Supplementary Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

In vitro selected GUAA tetraloop-binding receptors with structural plasticity and evolvability towards natural RNA structural modules

Zakrevsky

Calkins

Kao

et al. 2021

Nucleic Acids Research

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GNRA tetraloop-binding receptor interactions are key components in the macromolecular assembly of a variety of functional RNAs. In nature, there is an apparent bias for GAAA/11nt receptor and GYRA/helix interactions, with the former interaction being thermodynamically more stable than the latter. While past in vitro selections allowed isolation of novel GGAA and GUGA receptors, we report herein an in vitro selection that revealed several novel classes of specific GUAA receptors with binding affinities comparable to those from natural GAAA/11nt interactions. These GUAA receptors have structural homology with double-locked bulge RNA modules naturally occurring in ribosomal RNAs. They display mutational robustness that enables exploration of the sequence/phenotypic space associated to GNRA/receptor interactions through epistasis. Their thermodynamic self-assembly fitness landscape is characterized by a rugged neutral network with possible evolutionary trajectories toward natural GNRA/receptor interactions. High throughput sequencing analysis revealed synergetic mutations located away from the tertiary interactions that positively contribute to assembly fitness. Our study suggests that the repertoire of GNRA/receptor interactions is much larger than initially thought from the analysis of natural stable RNA molecules and also provides clues for their evolution towards natural GNRA/receptors.

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“…Sequencing reads were processed using trimmomatic SE CROP:90 to facilitate joining 76 , and then paired-end reads were joined and unique sequences were enumerated using EasyDIVER 77 . Joining was performed using the following PANDAseq 78 flags: -a -l 1 -A pear -C completely_miss_the_point:0.…”

Section: Computational Analyses Of K-seq Datamentioning

confidence: 99%

“…Sequences were clustered into families based on sequence similarity, using a custom Python script (see Data Availability). The script ClusterBOSS.py uses the enumerated read output files generated from the EasyDIVER package 77 . In general, first, all sequences were sorted according to their read count values.…”

Section: Clustering Analysis Of Sequences From Selectionsmentioning

confidence: 99%

Error minimization and specificity could emerge in a genetic code as by-products of prebiotic evolution

Janzen

Shen

Liu

et al. 2021

Preprint

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The emergence of the genetic code was a major transition in the evolution from a prebiotic RNA world to the earliest modern cells. A prominent feature of the standard genetic code is error minimization, or the tendency of mutations to be unusually conservative in preserving biophysical features of the amino acid. While error minimization is often assumed to result from natural selection, it has also been speculated that error minimization may be a by-product of emergence of the genetic code. During establishment of the genetic code in an RNA world, self-aminoacylating ribozymes would enforce the mapping of amino acids to anticodons. Here we show that expansion of the genetic code, through co-option of ribozymes for new substrates, could result in error minimization as an emergent property. Using self-aminoacylating ribozymes previously identified during an exhaustive search of sequence space, we measured the activity of thousands of candidate ribozymes on alternative substrates (activated analogs for tryptophan, phenylalanine, leucine, isoleucine, valine, and methionine). Related ribozymes exhibited preferences for biophysically similar substrates, indicating that co-option of existing ribozymes to adopt additional amino acids into the genetic code would itself lead to error minimization. Furthermore, ribozyme activity was positively correlated with specificity, indicating that selection for increased activity would also lead to increased specificity. These results demonstrate that by-products of the evolution and functional expansion of a ribozyme system could lead to adaptive properties of a genetic code. Such 'spandrels' could thus underlie significant prebiotic developments.

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EasyDIVER: A Pipeline for Assembling and Counting High-Throughput Sequencing Data from In Vitro Evolution of Nucleic Acids or Peptides

Cited by 15 publications

References 13 publications

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

In vitro selected GUAA tetraloop-binding receptors with structural plasticity and evolvability towards natural RNA structural modules

Error minimization and specificity could emerge in a genetic code as by-products of prebiotic evolution

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