Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding the influence of mutations in the SARS-CoV-2 gene on clinical outcomes is critical for treatment and prevention. Here, we analyzed all high-coverage complete SARS-CoV-2 sequences from GISAID database from January 1, 2020, to January 1, 2021, to mine the mutation hotspots associated with clinical outcome and developed a model to predict the clinical outcome in different epidemic strains. Exploring the cause of mutation based on RNA-dependent RNA polymerase (RdRp) and RNA-editing enzyme, mutation was more likely to occur in severe and mild cases than in asymptomatic cases, especially A > G, C > T, and G > A mutations. The mutations associated with asymptomatic outcome were mainly in open reading frame 1ab (ORF1ab) and N genes; especially R6997P and V30L mutations occurred together and were correlated with asymptomatic outcome with high prevalence. D614G, Q57H, and S194L mutations were correlated with mild and severe outcome with high prevalence. Interestingly, the single-nucleotide variant (SNV) frequency was higher with high percentage of nt14408 mutation in RdRp in severe cases. The expression of ADAR and APOBEC was associated with clinical outcome. The model has shown that the asymptomatic percentage has increased over time, while there is high symptomatic percentage in Alpha, Beta, and Gamma. These findings suggest that mutation in the SARS-CoV-2 genome may have a direct association with clinical outcomes and pandemic. Our result and model are helpful to predict the prevalence of epidemic strains and to further study the mechanism of mutation causing severe disease.

Species identification of oaks (Quercus) is always a challenge because many species exhibit variable phenotypes that overlap with other species. Oaks are notorious for interspecific hybridization and introgression, and complex speciation patterns involving incomplete lineage sorting. Therefore, accurately identifying Quercus species barcodes has been unsuccessful. In this study, we used chloroplast genome sequence data to identify molecular markers for oak species identification. Using next generation sequencing methods, we sequenced 14 chloroplast genomes of Quercus species in this study and added 10 additional chloroplast genome sequences from GenBank to develop a DNA barcode for oaks. Chloroplast genome sequence divergence was low. We identified four mutation hotspots as candidate Quercus DNA barcodes; two intergenic regions (matK-trnK-rps16 and trnR-atpA) were located in the large single copy region, and two coding regions (ndhF and ycf1b) were located in the small single copy region. The standard plant DNA barcode (rbcL and matK) had lower variability than that of the newly identified markers. Our data provide complete chloroplast genome sequences that improve the phylogenetic resolution and species level discrimination of Quercus. This study demonstrates that the complete chloroplast genome can substantially increase species discriminatory power and resolve phylogenetic relationships in plants.

BACKGROUND: Primary hypertrophic osteoarthropathy (PHO) is a genetically and clinically heterogeneous systematic disorder caused by mutations in genes HPGD and SLCO2A1. The purpose of the present study is to provide useful information for the early and precise diagnosis of PHO and identify causative mutations in Chinese PHO children.
RESULTS: The clinical manifestations, radiographic features of seven Chinese pediatric patients were systematically analyzed. Targeted exome sequencing identified a previously reported c.310_311delCT mutation and a novel common splicing site mutation c.324 + 5G > A in the HPGD gene. Relative quantitative real time PCR validated a novel deletion of the exon 4 in the same gene. Neither mutations nor structural variations in the gene SLCO2A1 were detected.
CONCLUSIONS: In the present study, homozygous or compound heterozygous HPGD mutations were identified in seven Chinese pediatric patients, suggesting an autosomal recessive inheritance. The c.310_311delCT mutation and the splicing site mutation c.324 + 5G > A were likely to be mutational hotspots in Chinese PHO patients. For the first time, a structural variation of the HPGD gene was reported. Homozygous, compound heterozygous mutations or structural variation identified in the HPGD gene proposed that targeted exome sequencing may be a preferable method for pediatric PHO diagnosis and mutation analysis.

Herbaceous bamboos (Olyreae) are a separate lineage with idiosyncratic traits, e.g., unisexual flowers and annual or seasonal flowering lifestyle, in the grass family. To elucidate the evolution of herbaceous bamboos we produced two complete chloroplast (cp) genomes from two monotypic genera i.e., Froesiochloa and Rehia via the genome-skimming approach. The assembled F. boutelouoides and R. nervata cp genomes were 135,905 and 136,700 base-pair (bp), respectively. Further whole-genome comparative analyses revealed that the cp genes order was perfectly collinear, but the inverted repeats (IRs) borders, i.e., the junctions between IRs and single copy regions, were highly divergent in Olyreae. The IRs expansions/contractions occurred frequently in Olyreae, which have caused gene content and genome size variations, e.g., the copy number reduction of rps19 and trnH(GUG) genes in Froesiochloa. Subsequent nucleotide mutation analyses uncovered a greatly heterogeneous divergence pattern among different cpDNA regions in Olyreae cp genomes. On average, non-coding loci evolved at a rate of circa 1.9 times faster than coding loci, from which 20 rapidly evolving loci were determined as potential genetic markers for further studies on Olyreae. In addition, the phylogenomic analyses from 67 grass plastomes strongly supported the phylogenetic positions of Froesiochloa and Rehia in the Olyreae.

Eucommia ulmoides (E. ulmoides), the sole species of Eucommiaceae with high importance of medicinal and industrial values, is a Tertiary relic plant that is endemic to China. However, the population genetics study of E. ulmoides lags far behind largely due to the scarcity of genomic data. In this study, one complete chloroplast (cp) genome of E. ulmoides was generated via the genome skimming approach and compared to another available E. ulmoides cp genome comprehensively at the genome scale. We found that the structure of the cp genome in E. ulmoides was highly consistent with genome size variation which might result from DNA repeat variations in the two E. ulmoides cp genomes. Heterogeneous sequence divergence patterns were revealed in different regions of the E. ulmoides cp genomes, with most (59 out of 75) of the detected SNPs (single nucleotide polymorphisms) located in the gene regions, whereas most (50 out of 80) of the indels (insertions/deletions) were distributed in the intergenic spacers. In addition, we also found that all the 40 putative coding-region-located SNPs were synonymous mutations. A total of 71 polymorphic cpDNA fragments were further identified, among which 20 loci were selected as potential molecular markers for subsequent population genetics studies of E. ulmoides. Moreover, eight polymorphic cpSSR loci were also developed. The sister relationship between E. ulmoides and Aucuba japonica in Garryales was also confirmed based on the cp phylogenomic analyses. Overall, this study will shed new light on the conservation genomics of this endangered plant in the future.