An integrative protocol for one‑step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.

Contribution of the SOS response and the DNA repair systems to norfloxacin induced mutations in E.coli

Antibiotic-resistant bacteria severely threaten human health.Besides spontaneous mutations generated by endogenous factors,the resistance might also originate from mutations induced by certain antibiotics,such as the fluoroquinolones.Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems.However,the relative contributions of these molecular mechanisms remain unclear,hindering understanding of the generation of resistant pathogens.Here,using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains,as well as those of the strains deficient for the mismatch repair(MMR)and the oxidative damage repair pathways,we find that the SOS response is the major mutagenesis contributor in mutation elevation,responsible for~30–50%of the total base-pair substitution(BPS)mutation-rate elevation upon treatment with sublethal levels of norfloxacin(0~50 ng/mL).We further estimate the significance of the effects on other mutational features of these mechanisms(i.e.,transversions,structural variations,and mutation spectrum)in E.coli using linear models.The SOS response plays a positive role in all three mutational features(mutation rates of BPSs,transversions,structural variations)and affects the mutational spectrum.The repair systems significantly reduce the BPS mutation rate and the transversion rate,regardless of whether antibiotics are present,while significantly increasing the structural variation rate in E.coli.Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.

The Xa7 resistance gene guards the rice susceptibility gene SWEET14 against exploitation by the bacterial blight pathogen

Many plant disease resistance(R)genes function specifically in reaction to the presence of cognate effectors from a pathogen.Xanthomonas oryzae pathovar oryzae(Xoo)uses transcription activator-like effectors(TALes)to target specific rice genes for expression,thereby promoting host susceptibility to bacterial blight.Here,we report the molecular characterization of Xa7,the cognate R gene to the TALes AvrXa7 and PthXo3,which target the rice major susceptibility gene SWEET14.Xa7 was mapped to a unique 74-kb region.Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element(EBE)in its promoter and encoded a 113-amino-acid peptide of unknown function.Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains.Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana.The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility.Ectopic expression of Xa7 triggers cell death in N.benthamiana.Xa7 is prevalent in indica rice accessions from 3000 rice genomes.Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.