TY - JOUR
T1 - Best Practices in Microbial Experimental Evolution
T2 - Using Reporters and Long-Read Sequencing to Identify Copy Number Variation in Experimental Evolution
AU - Spealman, Pieter
AU - De, Titir
AU - Chuong, Julie N.
AU - Gresham, David
N1 - Publisher Copyright:
© 2023, The Author(s).
PY - 2023/6
Y1 - 2023/6
N2 - Copy number variants (CNVs), comprising gene amplifications and deletions, are a pervasive class of heritable variation. CNVs play a key role in rapid adaptation in both natural, and experimental, evolution. However, despite the advent of new DNA sequencing technologies, detection and quantification of CNVs in heterogeneous populations has remained challenging. Here, we summarize recent advances in the use of CNV reporters that provide a facile means of quantifying de novo CNVs at a specific locus in the genome, and nanopore sequencing, for resolving the often complex structures of CNVs. We provide guidance for the engineering and analysis of CNV reporters and practical guidelines for single-cell analysis of CNVs using flow cytometry. We summarize recent advances in nanopore sequencing, discuss the utility of this technology, and provide guidance for the bioinformatic analysis of these data to define the molecular structure of CNVs. The combination of reporter systems for tracking and isolating CNV lineages and long-read DNA sequencing for characterizing CNV structures enables unprecedented resolution of the mechanisms by which CNVs are generated and their evolutionary dynamics.
AB - Copy number variants (CNVs), comprising gene amplifications and deletions, are a pervasive class of heritable variation. CNVs play a key role in rapid adaptation in both natural, and experimental, evolution. However, despite the advent of new DNA sequencing technologies, detection and quantification of CNVs in heterogeneous populations has remained challenging. Here, we summarize recent advances in the use of CNV reporters that provide a facile means of quantifying de novo CNVs at a specific locus in the genome, and nanopore sequencing, for resolving the often complex structures of CNVs. We provide guidance for the engineering and analysis of CNV reporters and practical guidelines for single-cell analysis of CNVs using flow cytometry. We summarize recent advances in nanopore sequencing, discuss the utility of this technology, and provide guidance for the bioinformatic analysis of these data to define the molecular structure of CNVs. The combination of reporter systems for tracking and isolating CNV lineages and long-read DNA sequencing for characterizing CNV structures enables unprecedented resolution of the mechanisms by which CNVs are generated and their evolutionary dynamics.
KW - Copy-number variant
KW - Fluorescent CNV reporter
KW - Genome dynamics
KW - Long term experimental evolution
KW - Long-read sequencing
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U2 - 10.1007/s00239-023-10102-7
DO - 10.1007/s00239-023-10102-7
M3 - Review article
C2 - 37012421
AN - SCOPUS:85148436931
SN - 0022-2844
VL - 91
SP - 356
EP - 368
JO - Journal of Molecular Evolution
JF - Journal of Molecular Evolution
IS - 3
ER -