Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene

Abstract

Certain plants increase their freezing tolerance in response to low nonfreezing temperatures, an adaptive process named cold acclimation. Light has been shown to be required for full cold acclimation, although how light and cold signals integrate and cross-talk to enhance freezing tolerance still remains poorly understood. Here, we show that HY5 levels are regulated by low temperature transcriptionally, via a CBF-and ABA-independent pathway, and posttranslationally, via protein stabilization through nuclear depletion of COP1. Furthermore, we demonstrate that HY5 positively regulates cold-induced gene expression through the Z-box and other cisacting elements, ensuring the complete development of cold acclimation. These findings uncover unexpected functions for HY5, COP1, and the Z-box in Arabidopsis response to low temperature, provide insights on how cold and light signals integrate to optimize plant survival under freezing temperatures, and reveal the complexity of the molecular mechanisms plants have evolved to respond and adapt to their fluctuating natural environment.

The role of replicates for error mitigation in next-generation sequencing

“…NGS technologies are providing insights into genetic disease associations 57–63 , differences in human gut microbiota 64 , amino acid essentiality in proteins 65 , experimental evolution 66–68 , biotherapeutic development 69–73 , protein-DNA interactions 74 , epigenetics 75 , cancer genomics 39,76 and clinical diagnosis 77 . Efforts to find biologically and clinically relevant variants are steadily improving as algorithmic advances more intelligently filter the large amounts of sequence data.…”

SEDS proteins are a widespread family of bacterial cell wall polymerases

“…Mut-seq was performed on two independently generated libraries, as previously described 29 . In brief, PCR mutagenesis of the rodA open reading frame was performed using GeneMorph II Random Mutagenesis Kit (Stratagene) under conditions yielding ~1.4 mut/kb with primers oDR1067 and oDR1068 and PY79 genomic DNA.…”

“…To test whether RodA was responsible for glycan strand polymerization, we sought to assay non-functional mutants. We screened for essential residues in RodA by mutagenesis followed by high-throughput sequencing (MutSeq) 29 . Among the residues identified in our screen (Supplementary Table 1 and Extended Data Fig.…”

Validation of Next Generation Sequencing Cancer Panels for Clinical Somatic Mutation Profiling-- Identification of Source of Variations and Artifacts using FFPE Tissues

“…Whether or not another round of new sequencing technologies comes soon to replace current "next generation" sequencing technologies, their revolutionary impact is here to stay. Recently a huge impulse of NGS publications flooded into the scientific journals [1][2][3][4][5][6][7][8][9][10][11][12]. This is understandable as scientists try to make their mark in the NGS field.…”